Topics in Fluorescence Spectroscopy Vol. 2 Principles

Topics in
Fluorescence Spectroscopy
Volume 2
Principles
Topics in Fluorescence Spectroscopy
Edited by JOSEPH R. LAKOWICZ
Volume 1: Techniques
Volume 2: Principles
Volume 3: Biochemical Applications
Topics in
Fluorescence
Spectroscopy
Volume 2
Principles
Edited by
JOSEPH R. LAKOWICZ
Center for Fluorescence Spectroscopy
Department of Biological Chemistry
University of Maryland School of Medicine
Baltimore, Maryland
KLUWER ACADEMIC PUBLISHERS

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Contributors
Katalin Ajtai • Department of Biochemistry and Molecular Biology, Mayo

Foundation, Rochester, Minnesota 55905; permanent address: Department of
Biochemistry, Eötvös Loránd University, Budapest, Hungary
Marcel Ameloot • Limburgs Universitair Centrum, Universitaire Campus,
B-3610 Diepenbeek, Belgium
Joseph M. Beechem • Department of Physics, University of Illinois
Urbana–Champaign, Urbana, Illinois 61801; present address: Department of
Molecular Physiology and Biophysics, Vanderbilt University, Nashville,
Tennessee 37232
Ludwig Brand • Department of Biology, The John Hopkins University,
Baltimore, Maryland 21218
Thomas P. Burghardt • Department of Biochemistry and Molecular Biology,
Mayo Foundation, Rochester, Minnesota 55905
Herbert C. Cheung • Department of Biochemistry, University of Alabama
at Birmingham, Birmingham, Alabama 35294
Maurice R. Eftink • Department of Chemistry, University of Mississippi,
University, Mississippi 38677
Susan G. Frasier-Cadoret • Departments of Pharmacology and Internal
Medicine, University of Virginia Health Sciences Center, Charlottesville,
Virginia 22908; present address: Office of Interdisciplinary Graduate Studies,
University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Enrico Gratton • Department of Physics, University of Illinois Urbana–
Champaign, Urbana, Illinois 61801
Michael L. Johnson • Departments of Pharmacology and Internal
Medicine, University of Virginia Health Sciences Center, Charlottesville,
Virginia 22908
Jay R. Knutson • National Heart, Lung, and Blood Institute, National
Institutes of Health, Bethesda, Maryland 20892
v
vi Contributors
s
Nicolai A. Nemkovich • Institute of Physics of the B.S.S.R. Academy of

Sciences, Minsk 220602, U.S.S.R.
Anatolyi N. Rubinov • Institute of Physics of the B.S.S.R., Academy of
Robert F. Steiner • Department of Chemistry, University of Maryland–
Baltimore County, Baltimore, Maryland 21228
Martin Straume • Departments of Pharmacology and Internal Medicine,
University of Virginia Health Sciences Center, Charlottesville, Virginia 22908;
present address: Department of Biology, The Johns Hopkins University,
Baltimore, Maryland 21218
Richard B. Thompson • Department of Biological Chemistry, University of
Maryland School of Medicine, Baltimore, Maryland 21201
Vladimir I. Tomin • Institute of Physics of the B.S.S.R., Academy of
Preface
Fluorescence spectroscopy and its applications to the physical and life sciences
have evolved rapidly during the past decade. The increased interest in
fluorescence appears to be due to advances in time resolution, methods of
data analysis, and improved instrumentation. With these advances, it is now
practical to perform time-resolved measurements with enough resolution to
compare the results with the structural and dynamic features of macro-
molecules, to probe the structures of proteins, membranes, and nucleic acids,
and to acquire two-dimensional microscopic images of chemical or protein
distributions in cell cultures. Advances in laser and detector technology have
also resulted in renewed interest in fluorescence for clinical and analytical
chemistry.
Because of these numerous developments and the rapid appearance of
new methods, it has become difficult to remain current on the science of
fluorescence and its many applications. Consequently, I have asked the
experts in particular areas of fluorescence to summarize their knowledge and
the current state of the art. This has resulted in the initial two volumes of
Topics in Fluorescence Spectroscopy, which is intended to be an ongoing
series which summarizes, in one location, the vast literature on fluorescence
spectroscopy. The third volume will appear shortly.
The first three volumes are designed to serve as an advanced text. These
volumes describe the more recent techniques and technologies (Volume 1),
the principles governing fluorescence and the experimental observables
(Volume 2), and applications in biochemistry and biophysics (Volume 3).
Additional volumes will be published as warranted by further advances in
this field. I welcome your suggestions for future topics or volumes, offers to
contribute chapters on specific topics, or comments on the present volumes.
Finally, I thank all the authors for their patience with the delays incurred
in release of the first three volumes.
Joseph R. Lakowicz
Baltimore, Maryland
vii
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Contents
1. Fluorescence Anisotropy: Theory and Applications

Robert F. Steiner
1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2. Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.1. Meaning of Anisotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2. Influence of Excitation Pulse Shape. . . . . . . . . . . . . . . . . . . . . 5
1.2.3. The Time Decay of Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.4. The Rotational Diffusion of Ellipsoids of Revolution. . . . . . . . 8
1.2.5. The Anisotropy Decay of Ellipsoidal Particles . . . . . . . . . . . . 10
1.2.6. Partially Immobilized Systems . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.2.7. The Influence of Internal R o t a t i o n . . . . . . . . . . . . . . . . . . . . . . 18
1.3. Experimental Analysis of Anisotropy Decay . . . . . . . . . . . . . . . . . . . 22
1.3.1. Analysis of Time-Domain Data . . . . . . . . . . . . . . . . . . . . . . . . 22
1.3.2. Time-Domain Measurements of Anisotropy Decay. . . . . . . . . . 25
1.3.3. Frequency-Domain Measurements of Anisotropy Decay. . . . 26
1.4. Anisotropy Decay of Heterogeneous Systems. . . . . . . . . . . . . . . . . . . . . 28
1.4.1. Anisotropy-Resolved Emission Spectra. . . . . . . . . . . . . . . . . . . . . 28
1.4.2. The Meaning of Correlation Times for Associative and
Nonassociative Heterogeneity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
1.5. Anisotropy Decay of Intrinsic Protein Fluorophores................ 32
1.5.1. Anisotropy Decay of a Rigid Protein: S. Nuclease........... 32
1.5.2. Rotational Dynamics of Flexible Polypeptides: Adreno-
corticotropin and Melittin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
1.5.3. Anisotropy Decay of a Tightly Bound Fluorophore:
Lumazine Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
1.5.4. Anisotropy Decay of a Transfer RNA . . . . . . . . . . . . . . . . . . . 39
1.6. Anisotropy Decay of Biopolymers Labeled with an Extrinsic
Fluorophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
1.6.1. Anisotropy Decay and Internal Flexibility of Myosin . . . . . . 41
1.6.2. Anisotropy Decay of a Fibrous Protein: F-Actin.......... 43
1.6.3. Anisotropy Decay for Proteins Displaying Internal Rotation
Involving a Well-Defined Domain: The Immunoglobulins.. 44
ix
x Contents
1.6.4. Anisotropy Decay of Calmodulin Complexes with TNS . . . . 48

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2. Fluorescence Quenching: Theory and Applications

Maurice R. Eftink
2.1. Introduction ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.2. Basic Concepts.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.2.1. The Stern–Volmer Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.2.2. Quenching Mechanisms and Efficiency . . . . . . . . . . . . . . . . . . 58
2.2.3. Diffusional Nature of Quenching . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2.4. Static Quenching. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
2.2.5. Various Quenchers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
2.3. Quenching Studies with Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
2.3.1. Exposure of Fluorophores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
2.3.2. Effect of the Macromolecule’s Size . . . . . . . . . . . . . . . . . . . . . . 68
2.3.3. Electrostatic Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2.3.4. Tryptophan Residues in Proteins . . . . . . . . . . . . . . . . . . . . . . . 72
2.3.5. Ligand Binding and Conformational Changes . . . . . . . . . . . . 75
2.3.6. Mechanism of Quenching in Proteins—Penetration versus
Unfolding Mechanisms .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.3.7. Interaction of Quenchers with Proteins. . . . . . . . . . . . . . . . . . . . . 85
2.3.8. Transient Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
2.3.9. Multiple Quenching Rate Constants and Fluorescence
Lifetimes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.4. Studies with Membranes and Nucleic Acids. . . . . . . . . . . . . . . . . . . . . 92
2.4.1. Partitioning of Quenchers into Membranes/Micelles...... 92
2.4.2. Two-Dimensional Diffusion in Membranes. . . . . . . . . . . . . . . . 96
2.4.3. Quencher Moieties Attached to Lipid Molecules. . . . . . . . . . . . . 97
2.4.4. Membrane Transport and Surface Potential. . . . . . . . . . . . . . . . . 99
2.4.5. Nucleic Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
2.5. Uses to Resolve Other Fluorescence Properties. . . . . . . . . . . . . . . . . 101
2.5.1. Resolution of Steady-State Spectra. . . . . . . . . . . . . . . . . . . . . . . . 102
2.5.2. Resolution of Fluorescence Lifetimes. . . . . . . . . . . . . . . . . . . . . . 103
2.5.3. Resolution of Anisotropy Measurements. . . . . . . . . . . . . . . . . . . . . 105
2.5.4. Resolution of Energy Transfer Experiments . . . . . . . . . . . . . . . 108
2.5.5. Other Uses of Solute Quenching. . . . . . . . . . . . . . . . . . . . . . . . 109
2.6. Recent Developments in Data Analysis . . . . . . . . . . . . . . . . . . . . . . . 112
2.6.1. Simultaneous Analyses of Quenching D a t a . . . . . . . . . . . . . . . 112
2.6.2. Nonlinear Least-Squares Fits . . . . . . . . . . . . . . . . . . . . . . . . . . 113
2.6.3. Distribution of Lifetimes or Rate Constants . . . . . . . . . . . . . . 114
2.6.4. Experimental Improvements . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Contents xi
2.7. Phosphorescence Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

2.8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3. Resonance Energy Transfer

Herbert C. Cheung
3.1. Long-Range Dipole–Dipole Interaction. . . . . . . . . . . . . . . . . . . . . . . . . 128
3.2. Determination of Energy Transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
3.3. Proximity Mapping of Molecular Assembly . . . . . . . . . . . . . . . . . . 132
3.4. Experimental Strategy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.4.1. Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.4.2. Measurement of Transfer Efficiency. . . . . . . . . . . . . . . . . . . . . . . 133
3.4.3. The Orientation Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
3.5. Selected Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.5.1. Myosin and Actomyosin.................................... 140
3.5.2. Troponin Subunits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.5.3. Ribosomal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
3.6. Comparison of FRET Results with Results from Other
Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
3.6.1. Comparison with Crystallographic Data. . . . . . . . . . . . . . . . . . 148
3.6.2. Comparison with Cross-Linking Data. . . . . . . . . . . . . . . . . . . 151
3.7. Application of FRET to Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . 152
3.8. Time-Resolved Energy Transfer M e a s u r e m e n t s . . . . . . . . . . . . . . 155
3.9. Distribution of Distances. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
3.9.1. Theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3.9.2. Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.10. Summary and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
References .............................................. 171
4. Least-Squares Analysis of Fluorescence Data

Martin Straume, Susan G. Frasier-Cadoret, and Michael L. Johnson
4.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
4.2. Basic Terminology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.3. Assumptions of Least-Squares Analysis . . . . . . . . . . . . . . . . . . . . . . . 181
4.4. Least-Squares Parameter Estimation Procedures. . . . . . . . . . . . . . . . . . 186
4.4.1. Modified Gauss–Newton Algorithm. . . . . . . . . . . . . . . . . . . . . . . 187
4.4.2. Nelder–Mead Simplex Algorithm. . . . . . . . . . . . . . . . . . . . . . . . . . 193
4.5. An Example of the Least-Squares Procedures—Collisional
Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
xii Contents
4.5.1.Example of the Gauss–Newton Procedure . . . . . . . . . . . . . . . 202

4.5.2.Example of the Nelder–Mead Simplex Procedure . . . . . . . . . 205
4.6. Joint Confidence Intervals—Estimation and Propagation. . . . . . 207
4.6.1. Asymptotic Standard Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
4.6.2. Linear Joint Confidence Intervals. . . . . . . . . . . . . . . . . . . . . . . . . . 208
4.6.3. Support Plane Confidence Intervals. . . . . . . . . . . . . . . . . . . . . . . . 209
4.6.4. Approximate Nonlinear Support Plane Joint Confidence
Intervals ........................................... 211
4.6.5. A Monte Carlo Method for the Evaluation of Confidence
Intervals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
4.6.6. Propagation of Confidence Intervals . . . . . . . . . . . . . . . . . . . . 215
4.7. Analysis of Residuals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
4.7.1. Plots. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
4.7.2. Distributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
4.7.3. Trends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
4.7.4. Outliers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
4.7.5. Influential Observations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
4.7.6. Common Quantitative Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
4.8. Implementation Notes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
4.9. In Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
5. The Global Analysis of Fluorescence Intensity and Anisotropy Decay

Data: Second-Generation Theory and Programs
Joseph M. Beechem, Enrico Gratton, Marcel Ameloot, Jay R. Knutson,
and Ludwig Brand
5.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
5.1.1. Multiexcitation/Multitemperature Studies of Anisotropic
Rotation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
5.1.2. Multiexcitation/Emission Wavelength Studies of Total
Intensity D a t a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
5.1.3. Double-Kinetic Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
5.2. The Global Analysis Philosophy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
5.2.1. Evolution of the Global Analysis Approach . . . . . . . . . . . . . . 244
5.2.2. Global Analysis Implementation Strategy. . . . . . . . . . . . . . . . . . . 248
5.3. General Elements of the Global Analysis Program. . . . . . . . . . . . . . . . . 249
5.3.1. Mapping to the Physical Observables . . . . . . . . . . . . . . . . . . . 252
5.3.2. Empirical Description of the Fluorescence Decay . . . . . . . . . 252
5.3.3. Compartmental Description of Photophysical Events. . . . . . . . 253
5.3.4. Overview of Nonlinear Minimization (The Basic Equations) 258
Contents xiii
5.4. In-Depth Flow Chart of a General-Purpose Global Analysis

Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
5.4.1. Overview of the Global Analysis Procedure . . . . . . . . . . . . . 259
5.4.2. Flow Chart for the LFD Global Analysis Program
“Global”. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
5.5. Case Studies of the Application of Global Analysis to Experimental
Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
5.5.1. Case Study of a Two-State Excited State Reaction . . . . . . . 272
5.5.2. Distributions of Distances and Energy Transfer Analysis... 277
5.6. Anisotropy Decay Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
5.6.1. General Equations and Experimental Linkages . . . . . . . . . . . 280
5.6.2. Changes in Anisotropy Data Collection Schemes. . . . . . . . . . . . 283
5.6.3. Associative versus Nonassociative Modeling of Anisotropy 283
5.6.4. Anisotropy Decay-Associated Spectra (ADAS) . . . . . . . . . . . 284
5.6.5. Multidye Global Anisotropy Decay Analysis . . . . . . . . . . . . . 285
5.6.6. Distributed Lifetimes and Distributed Rotational Correlation
Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
5.6.7. Multiexcitation Anisotropy Experiments. . . . . . . . . . . . . . . . . . . . . 286
5.6.8. Example of Distributed Rotations: Fluorophore Rotations
Gated by Packing Fluctuations in Lipid B i l a y e r s . . . . . . . . . . 287
5.7. Error Analysis and the Identifiability Problem . . . . . . . . . . . . . . . . . 288
5.7.1. The Identifiability Problem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
5.7.2. Identifiability Study Using Laplace Identifiability Analysis.. 291
5.7.3. Error Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
5.8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
References ............................................... 301
6. Fluorescence Polarization from Oriented Systems

Thomas P. Burghardt and Katalin Ajtai
6.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
6.2. Theory and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
6.2.1. The Angular Probability Density N........................... 309
6.2.2. Fluorescence Polarization in Homogeneous Space . . . . . . . . 311
6.2.3. Time-Resolved Fluorescence Depolarization Determination
of the High-Resolution Angular Probability Density......... 320
6.2.4. Relation of Electron Spin Resonance Spectra to Fluorescence
Polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
6.2.5. Biochemical Techniques of Specific Labeling . . . . . . . . . . . . . 332
6.3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
xiv Contents
7. Fluorescence-Based Fiber-Optic Sensors

Richard B. Thompson
7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
7.2. Fiber-Optic Fundamentals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
7.3. Sensor Design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
7.4. Sensing Tip Configurations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
7.5. Fiber Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
7.6. Separating Excitation and Emission . . . . . . . . . . . . . . . . . . . . . . . . . 357
7.7. Launching Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
7.8. Light Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
7.9. Time-Resolved Fluorescence in Fibers . . . . . . . . . . . . . . . . . . . . . . 361
7.10. Polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
7.11. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
8. Inhomogeneous Broadening of Electronic Spectra of Dye Molecules in

Solutions
Nicolai A. Nemkovich, Anatolyi N. Rubinov, and Vladimir I. Tomin
8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
8.2. Theoretical Considerations of Inhomogeneous Broadening......... 369
8.2.1. Solvate Configurational Energy . . . . . . . . . . . . . . . . . . . . . . . . 369
8.2.2. Field Diagram of a Polar Solution. . . . . . . . . . . . . . . . . . . . . . . . . 371
8.2.3. Solvate Distribution in Configurational Sublevels . . . . . . . . . 374
8.2.4. Nonpolar Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
8.2.5. Selective Excitation with Vibrational Spectral Broadening.. 378
8.2.6. Absorption and Fluorescence Spectra: Dependence on
Exciting Light Frequency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
8.3. Stationary Inhomogeneous Broadening . . . . . . . . . . . . . . . . . . . . . . . . . . 387
8.3.1. Universal Relationship between Fluorescence and Absorp-
tion Spectra of Polar Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
8.3.2. Luminescence Spectra at Red-Edge Excitation. . . . . . . . . . . . . . . 388
8.3.3. Directed Nonradiative Energy Transfer in Organic Solutions 390
8.4. Dynamic Inhomogeneous Broadening in Liquid Solutions . . . . . . . 395
8.4.1. Analysis of Configurational Relaxation in Liquid Solutions 395
8.4.2. Experimental Study of the Luminescence Kinetics of Liquid
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
8.4.3. The Solution Spectrochronogram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
8.4.4. The Effect of Light-Induced Molecular Rotation in
Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
Contents xv
8.5. Selective Kinetic Spectroscopy of Fluorescent Molecules in

Phospholipid Membranes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
8.5.1. Energy Levels of an Electric Dipole Probe in a Membrane 413
8.5.2. Inhomogeneous Broadening in Steady-State Fluorescence
Spectra of Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
8.5.3. Kinetics of Probe Fluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
8.5.4. Rotational Dynamics of the Probe in the Membrane . . . . . . 422
8.6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
!"#$%&'()%#*+)*+#,*'--.%-)/+%0-'*1
1
Fluorescence Anisotropy:
Theory and Applications
Robert F. Steiner
1.1. Introduction
Fluorescence anisotropy decay belongs to the general class of relaxation

methods which monitor the time dependence of the transition of the system
from a biased to a random arrangement. In this case the transition is from
a specific to a random orientation in space and occurs via Brownian rota-
tional diffusion. Unlike some other techniques of this type, such as orientation
by the application of an external force field, fluorescence anisotropy decay
depends upon the initial selection of a population of specifically oriented
fluorophores from a large assembly of randomly oriented fluorophores.
From the nature of the change in anisotropy with time, information may be
derived as to the rotational mobility of the fluorophore. If the fluorophore is
a fluorescent label linked to a biological macromolecule it may reflect both
the overall motion of the particle and any internal rotational modes which
may be present, including the localized motion of the probe.(4)
Two basic kinds of information may be derived from fluorescence
anisotropy decay measurements. To the extent that the macromolecule and
the attached label rotate as a unit, anisotropy decay may provide information
as to the size and shape factor of the macromolecule. It is also a convenient
means of studying any internal rotational motions present in the macro-
molecule and of examining the nature of the molecular flexibility.
The basic theory of the depolarization of fluorescence through Brownian
rotation was presented by Perrin. Equations were developed which related
the degree of polarization to the size and shape parameters of a dissolved
fluorophore, the average lifetime of its excited state, and the temperature and
Robert F. Steiner • Department of Chemistry and Biochemistry, University of

Maryland–Baltimore County, Baltimore, Maryland 21228.
Topics in Fluorescence Spectroscopy, Volume 2: Principles, edited by Joseph R. Lakowicz. Plenum
Press, New York, 1991.
1
2 Robert F. Steiner
viscosity of the solvent. The theory of Perrin was tested using fluorophores of
known molecular volume and yielded reasonably self-consistent results.(6)
Subsequently, the use of the polarization of extrinsic fluorescent labels to
study proteins was introduced by Weber and applied to the characterization
of a number of proteins by Weber and others.(10, 11) The mathematical for-
mulation was substantially simplified by the adoption, first suggested by
Jablonski, of a linear parameter, the fluorescence anisotropy. (12) The use of
anisotropy is particularly advantageous for the study of heterogeneous
systems.
The development of the single-photon counting technique (13–15) made it
feasible to examine the time dependence of anisotropy directly. The basic
theory of anisotropy decay was described by Wahl(16, l7) and applied by Wahl
and co-workers to the study of the rotational dynamics of biopolymers(17–19).
The basic theory was expanded by Gottlieb and Wahl, (20) who con-
sidered the effects of internal rotations, and by Belford et al., who presented
a description of the anisotropy decay of ellipsoidal particles.(21) Kinosita et al.,
have extended the theory to the hindered rotation of fluorophores embedded
in membranes.(22)
The last decade has witnessed a progressive refinement of the technique
of time-domain measurements of anisotropy decay and the application of this
method to a wide variety of biopolymer and membrane systems. In recent
years frequency-domain determinations of anisotropy decay have been
developed as a useful alternative to time-domain measurements. The present
chapter will describe the current status of the technique and present some
selected applications.
1.2. Theory
1.2.1. Meaning of Anisotropy
The radiation emitted by a fluorophore may be polarized to varying

extents, depending upon conditions. The polarization is conventionally
characterized with reference to a system of laboratory coordinates defined by
the directions of observation and of the exciting beam. It is customary to
observe the fluorescence at an angle of 90° to the exciting beam (Figure 1.1).
If the center of the irradiated volume is chosen as the origin, O, and the x and
y axes are taken along the direction of observation and along the direction of
the exciting beam, respectively, then the directions Ox and Oy define a plane
containing all the instrumental elements. The Oz axis is perpendicular to this
plane.
The components of the total fluorescence intensity along the three coor-
Fluorescence Anisotropy: Theory and Applications 3
dinate axes are designated The sum of these three components

is equal to the total fluorescence intensity, S:
The exciting beam may correspond to either unpolarized, or natural, light or

to linearly polarized light whose direction of polarization is along the Ox or
Oz axis. In the case of unpolarized light, the electric vector may have any
orientation within the x-z plane. Symmetry considerations therefore require
that
so that the total intensity is given by, from Eq. (1.1),
When the exciting beam is polarized along the Oz axis, symmetry now
dictates that
and
Finally, if the exciting beam is linearly polarized along the Ox axis, we

have
and
4 Robert F. Steiner
It is usual to perform fluorescence anisotropy measurements with an

exciting beam which is polarized in the z-direction or, somewhat less com-
monly, with unpolarized light. For both cases, the measured quantities are the
components of fluorescence intensity which are polarized in the Oz and Oy
directions. (The component which is polarized in the Ox direction is not
accessible to measurement, as this is the direction of observation.) In this case
it is useful to define
and
In terms of the usual laboratory arrangement, may be identified

with the vertically and horizontally polarized components, respec-
tively.
The emission anisotropy, A, is defined by
where
Employing Eqs. (1.5) and (1.10), we obtain, for vertically polarized exciting
radiation,
and, for unpolarized exciting radiation,
It may be shown that the emission anisotropy for the two cases are related by
For horizontally polarized exciting light, we have
and
If more than one fluorescent species is present, the observed anisotropy is

given by (10)
where are the emission anisotropy of species i and its fractional

contribution to the total intensity, respectively.
An alternative formulation, which prevails in the older fluorescence
literature, employs polarization (P) rather than anisotropy. (10) The emission
polarization is defined by
However, since the use of anisotropy leads to simpler expressions, it has

replaced polarization in the recent literature and will be exclusively employed
in this chapter.
1.2.2. Influence of Excitation Pulse Shape
Observations of the time decay of fluorescence intensity or anisotropy

usually involve measurement of the summed response to a series of repetitive
light flashes.(23) If each excitation pulse were infinitely sharp, corresponding
to a function, and if only a single emitting species were present, then
the fluorescence intensity would decay with time according to a simple
exponential law:
where is the intensity at zero time, corresponding to the time of the

excitation pulse, t is the time after excitation, and is the average decay time
of fluorescence intensity.
It is at present feasible to obtain excitation pulses of picosecond width,
which approach the infinitely sharp case, by employing pulsed laser sources.(24)
However, the majority of anisotropy decay studies have made use of repetitive
spark discharges, whose widths are of the order of nanoseconds. In both cases
and especially for the latter case, decay and excitation processes overlap over
a finite time interval, so that it is not permissible to ignore the finite duration
of the excitation pulse.
The observed time decay profile i(t) may be related to the profile I(t)
6 Robert F. Steiner
which would be observed if the excitation pulse were infinitely sharp by the
convolution integral (25) :
Here, t is the time and E(u) is the time profile of excitation. (In this chapter
the convention will be followed of denoting experimentally observed quan-
tities, such as i, s, d, etc., by lowercase letters, while capital letters will be used
for the corresponding quantities I, S, D, etc., which have been corrected for
convolution effects so as to correspond to the behavior expected if the excita-
tion pulse were infinitely sharp.) The mathematical procedures which have
been developed for analyzing the time decay of fluorescence intensity in terms
of discrete decay times also provide for the deconvolution of the experimental
decay curve so as to remove the distortion caused by the finite duration of the
excitation pulse.
1.2.3. The Time Decay of Anisotropy
The measurement of fluorescence anisotropy depends upon the selective

and nonrandom excitation of fluorescent molecules or groups. When a
fluorophore absorbs a quantum of radiant energy, it undergoes a transition to
some vibrational level of a higher electronic state.(26) This is followed by a
very rapid (several picosecond) process of internal conversion which places
the fluorophore in a low vibrational level of the lowest electronic excited state.
This process generally attains completion prior to the emission of fluorescence.
It results in a nonequivalence of the transition moments corresponding to
absorption and emission. (1–3)
In the most general case the polarization properties of a fluorophore may
be described by a fourth-order tensor.(27) If the fluorophore is immobilized
in a rigid isotropic medium, so that Brownian rotation cannot occur, the
anisotropy, A0, is independent of time and is given by, for vertically polarized
excitation(1, 27, 28):
where are the components of the molecular absorption and

molecular emission tensors, along the three molecular axes.
For the case of common interest where absorption and emission are
strongly permitted electronic transitions and the wavelengths of measurement
correspond fairly closely to the 0–0 vibronic transition, the transition
moments of absorption and emission may be represented as linear with a well-
defined direction.(1) This model has proved adequate for the interpretation
of nearly all the available experimental data and will be assumed in the
discussion to follow. In this case,
and
where are the direction cosines of the linear transition moments of

absorption and emission, respectively, with respect to the molecular axes.
Equation (1.19) now becomes, if is the angle between the two linear
oscillators,
When a sample containing a population of fluorophores whose orienta-

tion is random is irradiated with vertically polarized light, a biased distribution
of orientations of the excited fluorophores is achieved by a photoselective
process. A fluorophore is excited with a probability which is proportional to
is the angle between the absorption transition moment and
the electric vector of the exciting beam. If the exciting beam is vertically
polarized, preferential excitation of those molecules whose transition moments
are oriented in the z-direction will occur. For unpolarized exciting radiation,
those molecules whose moments lie in the x–z plane are preferentially excited.
For short times after excitation, before significant Brownian rotation has
occurred, the relative magnitudes of reflect this biased distribution.
However, because of the finite breadth of the excitation pulse, Brownian
rotation occurs while excitation is still taking place, so that the maximum
observed value of A is less than the true value of For a fluorophore
with rotational mobility in a liquid medium, the direction of the transition
moments becomes progressively randomized with time until a uniform dis-
tribution is ultimately approached with and A =0 (Figure 1.2).
The time profile of anisotropy decay of a fluorophore is dependent upon
its rate of rotational diffusion. This is in turn related to its molecular charac-
teristics and, in the case of a fluorescent conjugate, to those of the biopolymer
to which it is linked. Most commonly, one is interested in the behavior of
a fluorescent probe joined covalently, or noncovalently, to a larger macro-
molecule. In order to obtain a manageable expression for the time dependence
of anisotropy, it is necessary to approximate the actual, somewhat irregular,
shape of the macromolecule by a smoothed geometrical form, such as an
ellipsoid of revolution.
We will consider first the case in which the fluorescent label is rigidly
8 Robert F. Steiner
attached to the macromolecule, with a fixed orientation of its transition

moments with respect to the coordinate axes of the latter. For a system of this
kind, the time decay of anisotropy depends not only upon the characteristics
of the macromolecule, but also upon the orientations of the transition
moments of absorption and emission with respect to the molecular axes.
Prior to describing the theory of anisotropy decay, it is appropriate to
discuss the rotational motion of rigid macromolecules, approximated as
ellipsoids.
1.2.4. The Rotational Diffusion of Ellipsoids of Revolution
The rate of rotational diffusion may be characterized by a rotary diffusion

constant, which is analogous to the familiar translational diffusion coefficient.
The rotational motion of a diameter of a rigid sphere with an arbitrarily
chosen orientation at zero time may be described by an equation analogous
to Fick’s second law:
Here, W is a probability density function describing the distribution of

rotational angles, w is the rotational angle of the spherical diameter, and D
is the rotary diffusion coefficient.
In the case of a spherical particle, the rotary diffusion coefficient, is
given by
where k is Boltzmann’s constant, T is the absolute temperature, is the

solvent viscosity, and V is the effective hydrodynamic volume, being equal to
the anhydrous volume plus an increment corresponding to the bound water
of hydration.
In order to describe the hydrodynamic properties of proteins, it is often

convenient to approximate their actual, somewhat irregular shape by a
smooth and symmetrical geometrical figure. Ellipsoids of revolution, which
are the three-dimensional bodies generated by rotating an ellipse about one of
its characteristic axes, are perhaps the most commonly used of these forms.
An elongated, or prolate, ellipsoid of revolution is generated by rotation
about the long axis of the ellipse, while a flattened, or oblate, ellipsoid of
revolution is formed by rotation about the short axis.
An ellipsoid has three principal axes, each of which is associated with a
characteristic rotational diffusion coefficient. The three diffusion coefficients
are designated where the subscript indicates the axis about
which diffusion occurs. In the case of a symmetrical ellipsoid of revolution, if
axis 1 corresponds to the axis of symmetry and axes 2 and 3 correspond to
the (equivalent) equatorial axes, then (Figure 1.3).
The rotary diffusion coefficients for rotation about the axis
of symmetry and the equatorial axes, respectively, may be related to the axial
ratio of the ellipsoid, and to the rotary diffusion coefficient, of the
equivalent sphere by, for a prolate ellipsoid of revolution(29):
It is also useful to define a set of three rotational correlation times,

which are functions of the rotational diffusion coefficients(30, 31):
10 Robert F. Steiner
For a particle with spherical symmetry,
Introducing the value of
1.2.5. The Anisotropy Decay of Ellipsoidal Particles
1.2.5.1. Theoretical Treatment of Anisotropy Decay

The time decay of anisotropy for a rigid ellipsoidal particle (not
necessarily an ellipsoid of revolution) has been treated by Belford et al.(21)
The model assumes that the fluorophore is immobilized with respect to the
particle, so that its transition moments have well-defined orientations with
respect to the axes of the particle. The time decay of anisotropy is in this
case governed by the magnitudes of the three rotational diffusion coefficients
corresponding to the three axes and by the directions of
the linear transition moments of absorption and emission. Belford et al. have
derived the following equation for the most general case:
Here, D (the mean rotational diffusion coefficient)
or 312) where are the cosines of the angles formed by the

transition moments of absorption with the three axes, and ' are
the corresponding direction cosines of the transition moments of emission.
The other quantities are defined by:
In the limiting case of a particle with spherical symmetry, for which

reduces to
Fluorescence Anisotropy Theory and Applications 11
where For a symmetrical ellipsoid of revolution, Eq. (1.29)

reduces to the sum of three exponentials:
Here,
where are the rotary diffusion coefficients for rotation about the
axis of symmetry and about either equatorial axis, respectively, and
where are the angles formed by the absorption and emission tran-
sition moments, respectively, with the axis of symmetry of the ellipsoid, and
is the angle formed by the projections of the two moments in the plane
perpendicular to the axis of symmetry.
For low values of corresponding to the initial slope of the
anisotropy decay curve (1) ,
Here, is the harmonic mean of the three correlation times and is defined,
in the general case, by:
For the special case where the transition moments are randomly oriented with
respect to the axes of the particle so that
then
In the case where the moments of absorption and emission coincide, then
so that Eqs. (1.36) become
For an ellipsoid of revolution for which the absorption or emission

moment is parallel to the axis of symmetry the time decay of
anisotropy is described by Eq. (1.33), but with replaced by
For the case of an ellipsoid of revolution for which the transition

moments of absorption and emission lie in a plane perpendicular to the axis
of symmetry, a more complex expression is obtained:
Here,
where is the rotational diffusion coefficient about the axis of symmetry,

and is the angle formed by the two transition moments.
In contrast to the first special case, the time decay of anisotropy is here
clearly nonexponential. Also, if the second term on the right-hand
side of Eq. (1.43) will be negative, leading to the possibility of negative
anisotropies at short times after excitation. Since, for a prolate ellipsoid of
revolution, is always greater than a transition to positive anisotropies
occurs at longer times in this case, followed by a monotonic decay to zero.
While, in principle, Eqs. (1.29) and (1.34) would permit the estimation of
the shape parameters of a rigid asymmetric protein whose shape can be
reasonably approximated by an ellipsoid, in practice instrumental limitations
normally make it difficult to monitor anisotropy decay over much more than
one decade. This is insufficient for the accurate detection of multiple correla-
tion times arising solely from molecular asymmetry. Further improvements in
instrumentation will probably be required before these relations can be
profitably capitalized on.
1.2.5.2. Simulation of Anisotropy Decay

An alternative approach to the time decay of fluorescence anisotropy has
been presented by Harvey and Cheung, who have utilized computer simula-
tion. (32) A large population of equivalent fluorescent molecules, each with its
own internal coordinates and orientations of the linear transition moments
of absorption and emission, is formally assembled by means of a random-
number generator, which is also used to produce the random stepwise motion
of Brownian rotation. The frame of reference of laboratory coordinates is
chosen so that the z axis is parallel to the direction of polarization of the
exciting beam, while the y and x axes lie in the direction of the exciting beam
and in that of observation, respectively. The angular distribution of orienta-
tions in the initial excited state is generated by setting the probability of
excitation proportional to is the angle formed by the
absorption transition moment with the z axis.
The normalized components of fluorescence intensity which are polarized
parallel and perpendicular to the direction of polarization of the
exciting beam are given by
where is the angle formed by the emission moment with the z axis.
The simulation of Brownian rotation was done by increasing the time in
a series of small increments and rotating the molecule at each step about
each of its axes by angular increments (/= 1, 2, 3) with the sign of
chosen randomly. The magnitude of each rotational step was determined by
the rotary diffusion coefficient about the corresponding axis, as predicted by
the classical theory of Brownian rotation:
After a set of time increments, the anisotropy was computed using Eqs.
(1.45)–(1.47). This was repeated for a series of such data sets, and the resultant
anisotropies were plotted as a function of time. This was done for oblate and
prolate ellipsoids of revolution with varying axial ratios and orientations of
the transition moments. In all cases the simulated anisotropy decay corre-
sponded closely to the predictions of the theory of Belford et al.(21)
A surprising prediction of the theory of Belford et al., which was verified
by the simulations of Harvey and Cheung, is that, for certain orientations of
the transition moments, the anisotropy actually increases with time for sort
intervals after excitation, so as to pass through a maximum before decaying
to zero at long times (Figure 1.4).
1.2.6. Partially Immobilized Systems
A special case of interest is that for which the rotational motion of a

labeled biopolymer is restricted so that free rotation occurs only about a single
axis. This might, for example, be the case for a labeled protein embedded in
a lipid bilayer which constrains its rotation to a single axis perpendicular to
the plane of the bilayer. In this case, rotational diffusion cannot depolarize the
fluorescence entirely, so that the anisotropy approaches a finite value at long
times. The time decay of anisotropy is given by, for a spherical particle(22, 34):
where is the correlation time for rotation of the particle about the fixed
axis, and The most general expression for the limiting
anisotropy at long times, is
where is the average value of the cosine squared for all possible angles
between the direction of the absorption transition moment at the time of
absorption and the emission transition moment at the time of emission.
If rotational wobble of the fluorophore is absent, so that rotation is
strictly confined to a single axis of the labeled biopolymer, then the motion
of the emission moment will be effectively confined to the surface of a cone of
semiangle is the angle formed by the emission dipole with the
direction of the axis about which rotation of the particle occurs. (1, 22, 34)
If the absorption and emission moments are parallel, then
Kinosita et al.(22) and Lipari and Szabo(34) have considered the parallel
case for which rotational wobble of the fluorophore is allowed. The rotating
unit, which may be either an unattached fluorescent probe or a labeled
biopolymer, is assumed to have cylindrical symmetry and the medium to have
uniaxial symmetry. The orientations of the transition dipoles of absorption
and emission with respect to the symmetry axis of the rotating unit are
assumed to be invariant, so that the equilibrium orientational distribution
of the two dipoles depends solely upon the angle between the axis of the
rotating unit and that of the medium (Figure 1.5). Since, for this case also, the
membrane-embedded fluorophore cannot assume all possible orientations
with equal probability, the anisotropy does not decay to zero at long times
but instead approaches a finite value,
A simple solution is possible if either the absorption dipole or the
emission dipole is parallel to the direction of the unique symmetry axis
of the rotating unit. If are unit vectors in the directions of the
absorption and emission dipoles and the axis of symmetry, respectively, then
A(t) may be expressed in terms of a correlation function as
Here, is the angle between P2 is the second Legendre polynomial

and the angle brackets indicate an equilibrium

average, defined as follows:
where is the equilibrium orientation distribution function. In Eq. (1.51)

the unit vector specifies the orientation of the probe at time t with respect
to a membrane-linked coordinate system (Figure 1.5).
In order to obtain an expression for the complete time dependence of
A(t), it is necessary to assume some model for the dynamics of the probe.
However, may be computed independently of any model:
and
where are the angles between the symmetry axis of the probe and
respectively.
Equation (1.54) relates the limiting fluorescence anisotropy to the order
parameter S of the probe, defined as
The order parameter governs the magnitude of the first nontrivial term in
the series expansion of the orientational distribution function in terms of
Legendre polynomials(34):
The order parameter thus furnishes model-independent information about the

orientational distribution function at equilibrium.
Kinosita et al. have developed an alternative formulation in terms of an
explicit model in which the probe can undergo free rotational diffusion within
a cone of semiangle This corresponds to the distribution function
For this model,
If either is parallel to then, using Eqs. (1.53) and (1.54),
To the extent that the diffusion in a cone model is valid, Eq. (1.59) permits
evaluation of the semiangle of the cone.
In order to evaluate the time-dependence of A(t) according to equation
(1.51), it is necessary to evaluate the correlation function
This requires the choice of a dynamic model. For the model corresponding to
diffusion in a cone, Kinosita et al. showed that (22)
where is the effective correlation time corresponding to the ith rotational

mode and is the corresponding amplitude. An approximate expression was
also derived in terms of a single rotational mode:
It may be shown (34) that is equal to the area under the curve of
1.2.7. The Influence of Internal Rotation
For real biopolymer systems, an intrinsic or extrinsic fluorescent label

which is rigidly integrated into the three-dimensional structure, so as to have
a fixed and well-defined orientation with respect to the coordinate axes of the
particle, is often not obtained. In many cases, some form of internal rotation
is present, so that a rotational motion of the label is superimposed upon that
of the entire particle.(20, 33, 34) The various types of internal rotation which are
possible may be roughly grouped into the following categories:
1. rotation of the fluorophore about the bond linking it to the

biopolymer;
2. rotational wobble of that portion of the biopolymer in proximity to
the fluorophore;
3. rotation of a well-defined molecular domain as a unit about a flexible
hinge point.
In practice, more than one rotational mode may be simultaneously present

(Figure 1.6).
The complexity of the general problem does not favor the development
of a comprehensive theory which would encompass a wide range of cases. In
particular, the data obtainable with the current instrumentation do not
readily lend themselves to analysis in terms of the more general theoretical
treatments, especially if more than one internal rotational mode is present.(35)
However, it is possible to obtain tractable solutions for several special cases
of interest.
In particular, if only a single internal rotational mode is present, then the
theoretical treatment outlined in Section 1.2.6 may be extended to the case of
a label attached to a large, freely rotating particle. If the assumptions cited
earlier are retained, then, for a spherical particle and a label which is confined
to a cone of semiangle
where are the correlation time of the particle and the effective
correlation time of the label, respectively, and is given by Eq. (1.59). If
then Eq. (1.62) reduces to the following relationship:
Equation (1.63) is usually applied in the more general form:
Here, depends solely on the localized rotation of the label, and reflects
the rotation of the entire macromolecule. The sum of is equal to the
anisotropy at zero time, A0.
For the case where the bond linking the fluorophore to the macro-
molecule has a sufficient degree of rotational wobble so that the fluorophore
may rotate freely within a cone, the magnitudes of a1 and a2 are given by an
equation analogous to Eq. (1.59)(22,34).
where is the semi-apex angle of the cone. Lipari and Szabo have shown
that Eq. (1.65) is valid for a probe with cylindrical symmetry, for which either
the absorption or emission moment is parallel to the cylindrical axis.(34) This
model implies the existence of a square-well potential which restricts rotation.
If wobble is absent, so that the emission moment is confined to the sur-
face of a cone, as would be the case if the emission moment makes a constant
angle with the axis of rotation, then we have
The preceding treatment is strictly valid only for the case when either
is parallel to the axis of the probe. If this is not the case, rotational
diffusion about the axis of the probe, as well as the rotational wobble of the
axis itself, can contribute to the time decay of anisotropy. The theory now
becomes considerably more complex. Lipari and Szabo have derived the
following approximate expression for the time dependence of anisotropy(34):
where are the respective angles between is

the difference between the azimuthal angles of are the
effective correlation times for rotation about the axis and for the rotation of
the axis itself, respectively. The quantities are reduced Wigner rotation
matrices:
In a subsequent theoretical development, Szabo has treated in detail a

number of models.(35)
Equation (1.64) is often loosely used as an empirical equation which has
been widely used to interpret anisotropy decay data. (4) In practice, use
of a two-term equation is usually compelled by the inability of available
techniques to reliably recover more than two correlation times.
In actual systems the existence of completely free rotation of the
fluorophore is somewhat improbable, and some degree of hindrance to
rotation is likely to be present. It is useful to consider the limiting case
where the label undergoes strongly hindered rotation. For this model the label
may exist in any of several orientations corresponding to potential energy
minima; the time spent in the specific positions is long in comparison with
the time spent in transit between positions. In this case the rate-limiting factor
for rotation is the probability of a jump between specific orientations. The
following equation was derived by Gottlieb and Wahl for this model(20):
where is the frequency of jumps between positions, K is a numerical

constant, and
Equation (1.69) may be rewritten in a form analogous to Eq. (1.64). The
parameters now have the meanings:
If the above model is strictly adhered to, w should be insensitive to

solvent viscosity, since it depends solely on the magnitude of the potential
energy barrier encountered by a transition between different positions. This
property may provide a potential means of ascertaining the validity of the
model. If the viscosity of the solvent is altered by the addition of sucrose,
glycerol, or some other viscosity-increasing substance, the value of is now
given by, from Eq. (1.70),
where is the factor by which the viscosity is increased. If measurements are

made for a series of viscosities and is linearly extrapolated versus to
then the intercept and slope should yield w and respectively. This
analysis is based on the assumption that the effective microviscosity sensed by
the label is equivalent to the bulk viscosity of the solvent. The rotating label
may be partially, or wholly, shielded from the solvent, so that the effective
microviscosity is less than the bulk viscosity. In this case, extrapolation
according to Eq. (1.71) would lead to an underestimation of and an over-
estimation of w.
The above simplified models do not adequately account for the more
general case where the independent motion of the probe is superimposed upon
the rotational wobble of the adjacent polypeptide or the rotation of a well-
defined subelement within the overall protein structure. With the usual
instrumentation available today, it is questionable whether more than two
correlation times can be reliably extracted from anisotropy decay data. If
more than two rotational modes are present, application of an equation of
the form of Eq. (1.64) will result in poorly defined averages. However, in
favorable cases where the probe is firmly immobilized in the tertiary structure,
the detection of a correlation time close to that expected for a well-defined
molecular domain is evidence for the free rotation of the domain.
1.3. Experimental Analysis of Anisotropy Decay
1.3.1. Analysis of Time-Domain Data
The analysis of single or multiexponential decay of anisotropy has most

commonly made use of the relationship
For vertically polarized exciting light,
where g is a factor correcting for the (usually imperfect) optical system and
is given by
where are the summed values over all times of respec-

tively, when the exciting light is horizontally polarized. In the absence of
distortion arising from grating effects, etc., should be equal. For the
determination of g, the fluorometer is normally operated in the static mode.
The time decay of fluorescence intensity may be represented by a model
of the form
where are the amplitude and decay time, respectively, of the ith
decay component.
The values of the may be determined from the experimental data
by a least-squares fitting procedure.(36) Trial values of the are used to
generate the intensity decay function S(t). For each set of the values
of the are obtained by solution of the corresponding set of equations linear
in the which are obtained for different times. The computed curve of
S(t) is convolved with the instrumental response function E(t), according

to Eq. (1.18), to yield a computed intensity decay function This is
compared with the experimental decay function and a value of the
criterion for goodness of fit, is calculated from
where are the values of respectively,

for the jth data point, and is the corresponding precision. Since single-
photon counting obeys Poisson statistics, is equal to where Y(j)
is the number of single-photon counts for the jth data point.
The set of values of the which correspond to a minimum value of
may be located by an iterative procedure in which each is incremented util
passes through a minimum. The value of corresponding to a minimal
may be located by parabolic interpolation. The process is repeated for each of
the in turn until an overall optimum fit is attained. For each set of values
of the the corresponding values of the are identified by a linear least-
squares fit.
The time-dependent fluorescence anisotropy, A(t), is given by
The time decay of anisotropy may be represented by a model of the form
Here, Ai and are the amplitude and rotational correlation time, respec-
tively, corresponding to the ith rotational mode, and is the limiting value
of the anisotropy attained at very long times after excitation. For fluorescent
molecules which are not partially, or wholly, immobilized in a matrix,
In evaluating the the previously computed values of the and
may be used to generate an impulse response function which represents
S(t), according to Eq. (1.77).(37) Trial values of the Ai and are used to
compute an analogous function for the anisotropy, A(t), according to
Eq. (1.79).(37) A(t) is then multiplied by S(t) to yield a trial representation of
the deconvoluted difference decay function D(t). D(t) is next convolved with
the time profile, E(t), of the excitation pulse, according to Eq. (1.18), to
generate a computed difference decay function is compared
with the experimental difference decay curve and a value of is
computed.
For each set of trial values of the Ai and the corresponding value of
is computed from
where are the values of these quantities for the jth data
point. The precision is taken as equal to where V(j) is the
associated variance. The optimum values of the Ai and are identified by an
iterative procedure similar to that used to determine the decay times.
The ultimate criterion of the quality of fit associated with a particular set
of parameters is the normalized value of This is equal to where
F is the number of degrees of freedom and is equal to the number of data
points (time channels) minus the number of determined parameters. For a
perfect fit, would have a value of unity.
A second useful criterion of quality of fit for both intensity and
anisotropy decay data is the distribution of residuals, that is, the difference
between observed and computed values. For an optimum fit the distribution
of residuals plotted as a function of time should be essentially random.
An alternative criterion of quality of fit is the Durbin–Watson parameter,
which is defined by (38)
The Durbin–Watson parameter monitors the correlation between the

residual values in neighboring channels. An optimum fit corresponds to a
maximum in this parameter, which implies a maximum randomness of
residuals.
An empirical procedure has proved to be useful in determining whether
is a real operating minimum for the set of optimized parameters.(39) A small
(2%) increase is made in the value of one parameter, and the corresponding
higher value of is computed. By parabolic extrapolation the parameter
value for a 5% increase in is determined. The procedure is repeated for a
2 % decrease in the value of the same parameter. If the spread in parameter
values is symmetrical, is an operating minimum. The process is repeated for
the other parameters in turn. The changes of 2% and 5% are of course
entirely arbitrary, but have proved satisfactory in practice.
1.3.2. Time-Domain Measurements of Anisotropy Decay
Time-domain measurements of anisotropy are generally made using a

single-photon-counting nanosecond fluorometer. (13–15) A pulsed light source
which flashes at a frequency of 15kHz to 2 MHz is used. Both lasers and
pulsed arc sources have been used. The electronic elements of instruments of
this kind must supply the following basic functions:
1. a start channel which times the beginning of a light pulse;

2. a stop channel which times the detection of single photons;
3. a selection channel which selects true single-photon events;
4. a data acquisition system whereby the accumulated photon counting
events are stored in the corresponding time channels in a multi-
channel analyzer.
The net result is the generation of a cumulative histogram of single-

photon counts stored in the time channels of the multichannel analyzer. This
is equivalent to a time profile of intensity. In order to avoid distortion of the
decay curve, precautions must be taken to avoid emission of more than one
photon as a consequence of a single lamp flash. In practice, this may be
achieved by limiting the rate of photon detection to about 2 % of that of lamp
flashing by a suitable adjustment of apertures.
Spectral selection for the excitation and emission beams may be achieved
with the use of either filters or gratings. The excitation beam is normally
vertically polarized for an anisotropy decay experiment. The orientation of the
excitation polarizer may be verified by comparing the intensities of light
scattered by a glycogen suspension with the emission analyzer in the vertical
and horizontal orientations. The latter intensity should be less than about 1 %
of the former.
The time profiles of intensity decay for the vertically and horizontally
polarized components of fluorescence intensity are accumulated and stored in
different sectors of the memory of the multichannel analyzer. In modern
instruments the emission analyzer is automatically alternated between vertical
and horizontal orientations for a series of short intervals, thereby minimizing
any differential effects of instrumental drift.
The time profile of the excitation is usually obtained using a scattering
solution, suxh as a Ludox silica suspension. Since fluorescence normally
occurs at a longer wavelength than scattered light, the wavelength dependence
of the photomultiplier may result in a significant time frame shift between the
fluorescence and reference scattering signals. This may be corrected for either
by determining the shift directly by measurement of samples of accurately
known lifetimes or by treating the shift as a variable whose value is chosen so
as to optimize the least-squares fit of intensity decay data.
1.3.3. Frequency-Domain Measurements of Anisotropy Decay
In recent years the measurement of anisotropy decay in the frequency

domain has been developed as an alternative to direct observation of the time
dependence. (40–43) In frequency-domain studies, the measured quantities are
the phase angle and the modulation m of fluorescence, which is excited with
light whose intensity i varies sinusoidally with time:
Here where f is the frequency. The modulation is defined by
For complete modulation, b = a and m = 1.

As a consequence of the finite duration of the excited state, the
modulated fluorescence emission is delayed in phase by an angle relative to
excitation. In addition, a decrease in modulation of the fluorescence occurs.
The intensity I of the fluorescence is given by
and the relative modulation, or demodulation factor, by
If only a single intensity decay mode is present, the phase and demodulation
factor are related to the decay time by
and
If multiple decay modes are present, the apparent values of computed from
Eqs. (1.86) and (1.87) will correspond to averages. Ih this case measurements
over a range of frequencies are necessary in order to obtain well-defined
physical parameters. The phase angle and modulation values may be pre-
dicted as a function of frequency for any postulated decay law. The assumed
decay law contains the information necessary for this prediction, just as it also
contains the information required to predict the directly observed time
dependence of fluorescence.
For frequency-domain measurements, sine and cosine transformations of
the assumed decay function (Eq. 1.77) are used instead of convolution with
the excitation lamp profile. If the quantities are defined by
then the phase angle and modulation may be predicted for each frequency
from
and
where are the computed values of the phase angle and demodula-
tion factor, respectively, for frequency
The quantities may be related directly to the intensity decay
parameters by
The goodness of fit between the computed and observed values of phase
angle and modulation may be assessed from the value of
Anisotropy decay may also be measured in the frequency domain. The

individual polarized components are related to the total intensity i by:
where
The quantities N and D may be evaluated for the vertically and horizon-
tally polarized components, using Eqs. (1.88) and (1.89). The quantities which
are actually measured are the phase angle difference between the two
components and the ratio of the modulated amplitudes:
The corresponding calculated values of respec-

tively) may be obtained using
and
The quality of fit is estimated from
where n is the number of degrees of freedom (the number of data points minus
the number of variable parameters), and are the estimated
uncertainties at frequency respectively.
In fitting actual data, trial values of the correlation times, are used to
compute the anisotropy as a function of time. The previously determined set
of intensity decay times, are used to compute the intensity, i(t), as a func-
tion of time. The substitution of A(t) and i(t) in Eqs. (1.95) yields computed
values of introduction of these into Eqs. (1.88) and (1.89)
provides values of as a function of frequency. Finally,
Eqs. (1.97) and (1.98) are used to generate trial values of
these are compared with the observed values, and a value of is calculated.
The set of assumed correlation times is varied systematically by an iterative
process until the set yielding a minimum value of is identified.
1.4. Anisotropy Decay of Heterogeneous Systems
1.4.1. Anisotropy-Resolved Emission Spectra
In a heterogeneous mixture of fluorescent species, fluorophores differing

in correlation time may also have different emission spectra.(44) A simple
example is a small fluorescent ligand which is reversibly bound by a large

biopolymer, such as a protein or nucleic acid. If the emission spectra of the
free and bound ligand are different, the observed static emission spectrum,
measured in the conventional way as the total intensity as a function of
wavelength, will be composite, corresponding to the sum of the individual
spectra. However, if instead of total intensity, S, the difference in intensity,
between the vertically and horizontally polarized components of
fluorescence is measured as a function of wavelength, the observed spectrum
will reflect primarily the complex species since D will be very small for the free
ligand. If D is measured as a function of wavelength for a series of times after
excitation, then only at very short times will the free ligand make a significant
contribution to the emission spectrum. At long times after excitation the
values of D for the free ligand will have decayed to zero, so that the observed
spectrum arises entirely from the complexed species. (For a homogeneous
system whose correlation time is long enough to permit measurement, the
spectra obtained using D and S are equivalent, since the wavelength
dependence of is the same as that for
More generally, for a heterogeneous system containing « species, the
anisotropy as a function of time and wavelength is given by (44)
Here, an overbar indicates a heterogeneous sum; represents the emission

spectrum of species i; and Di(t) and Si(t) are the values of D and S for
species i at time t. What is desired is the form of each The data required
are the time dependence of A, D, and S as a function of wavelength.
The necessary information is contained in
If is measured at n or more different times (t j ), the following set of

linear equations is obtained(38):
These correspond to the matrix equation

where each matrix element is given by
Further progress requires a knowledge of the individual functions

for each tj. These may be obtained through an analysis of the time decays of
anisotropy and intensity, as described in earlier sections. If each Di is known,
then the set of may be obtained by matrix inversion. The inverse of M,
G, is multiplied by the observed value of , The corresponding matrix
equation is
and
where Fi corresponds to the magnitude of the emission spectrum at wave-

length of component i.
In this way the correct linear combinations of the values of measured
at different times will yield the anisotropy decay-associated spectra of each
species, The method depends upon the obtaining of accurate values of
the Di. In some simple cases where a single decay time of fluorescence
intensity is unambiguously associated with a single rotational correlation time,
each individual Di is directly related to these quantities. Thus, for a binary
mixture whose species have distinct spectra, intensity decay times and
rotational correlation times we have
and
In more complex cases there may not be an obvious correspondence of

lifetimes and correlation times, and a direct analysis of in terms of a set of
decay times, each of which corresponds to a particular D i, may be
necessary.(44)
In practice, the above relationships must be modified to allow for
convolution effects and for the fact that time-resolved emission spectra are
generally measured over a range of time channels. In what follows, lower case
quantities will refer to the instrument (convolved) time scale. Thus, Di is

replaced by
where E(t) represents the excitation pulse (cf. Eq. (1.18)).

Equation (1.101) now becomes
where now refers to the jth time window, which extends from time
channel aj to bj for the jth time-resolved emission spectrum. Since convolution
affects solely the time axis, the matrix elements now are
Also,
where Gki is the inverse matrix of is the windowed difference time-

resolved emission spectrum, and is the desired anisotropy decay-
associated spectrum. The presence of convolution effects thus does not prevent
the extraction of anisotropy decay-associated spectra.
Davenport et al. have employed the preceding theory to resolve the
anisotropy decay-associated spectra of 1, 6-diphenyl-l, 3, 5-hexatriene (DPH)
which is distributed between vesicles of (DML) and
In this model system the DPH fluorophore
rotates rapidly within the DML vesicles, but rotates more slowly within the
DPL vesicles. The corresponding emission spectra are significantly different,
while DPH in a mixture of the two types of vesicles yields a static emission
spectrum of an intermediate type. Analysis of the mixed system using the
treatment described above yielded individual spectra which corresponded to
those obtained for purified vesicle systems.
1.4.2. The Meaning of Correlation Times for Associative and Nonassociative

Heterogeneity
Time-resolved decays of both intensity and anisotropy are often multi-

exponential. Both types of heterogeneity may be intrinsic to a single kind of
emitting species. A heterogeneity of intensity decay may arise from excited

state processes or from the existence of populations of distinct conformers;
a heterogeneity of anisotropy decay may reflect the presence of multiple
rotational modes, such as localized and global diffusion. Heterogeneity of the
above kinds, which is termed nonassociative, often permits a comparatively
straightforward interpretation of anisotropy. The anisotropy at a particular
time t is equal to is equal to A0, the limiting
anisotropy at zero time, which is characteristic of the fluorophore in the
absence of rotational diffusion. In this case any observed correlation modes
correspond to rotational modes involving the fluorophore of interest.
If the heterogeneity of intensity and anisotropy decay arises from multi-
ple emitting species, the situation is quite different. For heterogeneity of this
kind, which is termed associative, it is not usually possible to identify an
observed correlation time with a rotational mode of a particular fluorophore.
While the observed anisotropy at all times corresponds to the intensity-
weighted average of the anisotropies of the different species, according to
Eq. (1.15), the fractional intensities are themselves time-dependent (unless the
different emitting species have identical decay times). Thus, at long times after
excitation the observed average anisotropies become increasingly influenced
by the species of longest decay times.
In practice, a wide variety of anisotropy decay patterns, some of them
very bizarre, can arise in this way. For example, if two emitting species are
present, one of which has much longer decay times of both intensity and
anisotropy than the other, the anisotropy may actually pass through a mini-
mum and increase at longer times. In other, less dramatic cases, effects of this
kind may lead to plausible, but erroneous, interpretations of anisotropy
decay.
1.5. Anisotropy Decay of Intrinsic Protein Fluorophores
1.5.1. Anisotropy Decay of a Rigid Protein: S. Nuclease
Staphylococcus aureus nuclease B, whose molecular weight is 2.0 × 104,

contains a single tryptophan residue. Time-domain measurements of its
anisotropy decay were made in an early study by Munro et al. which
employed a synchroton light pulse as the excitation source.(4) The half-width
of this source, 0.65 ns, was substantially less than those of the flashlamps in
common use at that time and facilitated the detection of very rapid rotational
modes.
The time decay of fluorescence intensity at pH 7.4, 20 °C, was found to be
exponential, corresponding to a single decay time of 5.05 ns. The time decay
of anisotropy was also found to be exponential and could be described in

terms of a single rotational mode, of correlation time 9.85 ns. Thus, no
evidence for any internal rotational modes was found in this study.
Lakowicz et al. have subsequently reinvestigated this system, employing
frequency-domain measurements.(46) In contrast to the results of the earlier
study, two decay components were required for an optimal fit of the fluores-
cence intensity decay, with
respectively.
The time decay of fluorescence anisotropy was also biexponential, with
respectively. The con-
tribution of the more rapid rotational mode, although minor, appeared to be
real, in view of a twofold decrease in relative to that for the one-component
fit (Figure 1.7).
The model which emerges from these studies depicts the tryptophan as
sensing a relatively rigid microenvironment, in which its rotational motion is
confined to a rather narrow cone. Both studies assign a correlation time near
10 ns to the dominant rotational mode. The correlation time predicted for a
rigid hydrated sphere of the molecular weight of S. nuclease, assuming a
hydration of 0.2, is 7.6 ns. The somewhat larger value observed can easily be
attributed to the contribution of molecular asymmetry.
S. nuclease thus provides an example of a protein with only slight
mobility of its intrinsic fluorophore.
1.5.2. Rotational Dynamics of Flexible Polypeptides: Adrenocorticotropin and

Melittin
An example of the application of fluorescence anisotropy decay to a small

polypeptide with internal flexibility is provided by the study by Ross et al. of
adrenocorticotropin (ACTH).(47) The fluorescence of the single tryptophan
group of ACTH, Trp-9, was examined. The time decay of fluorescence
intensity could not be adequately fit by a single-exponential decay law. The
assumption of at least two decay times was required to yield an acceptable fit;
no further improvement occurred if three components were assumed. Two
decay components were found in the absence and presence of 20% sucrose
and at temperatures ranging from 3.5 °C to 15 °C (Table 1.1).
Since only one tryptophan is present, heterogeneity can be ruled out as
a source of the multiple decay components. As the decay times were inde-
pendent of emission wavelength, it is also unlikely that a two-state exited state
reaction is responsible. The most plausible explanation is in terms of a mobile
tryptophan group which can sample different microenvironments by rotation.
Contact with different amino acid side chains may result in different degrees
of quenching of the indole excited state and hence in multiple lifetimes.
This model was supported by measurements of the time decay of

anisotropy. For all conditions examined, a satisfactory fit was obtained only
with the assumption of two rotational modes (Table 1.2). The more rapid
rotational mode corresponded to a correlation time in the subnanosecond
range and was attributed to a localized motion of the tryptophan. The
magnitude of the shorter correlation time was almost independent of viscosity.
The longer correlation time, which was viscosity-dependent, had a magnitude
roughly consistent with rotation of the entire molecule.
The failure of the more rapid rotational mode to respond to an alteration
in viscosity is not unexpected if the rotation of the tryptophan involves a
transition between different potential energy minima and the rate-limiting step
is the probability of release from a position of minimum energy. Alternatively,
if the tryptophan rotates freely within a cone, the solvent composition and
effective viscosity in its vicinity may be different from those of the bulk
solution.
Lakowicz et al. have subsequently reexamined the ACTH system using
frequency-domain measurements.(46) There was qualitative agreement with the
time-domain results in that the time decays of both fluorescence intensity and
anisotropy were found to be biexponential (Table 1.1). As in the earlier study,
the anisotropy decay could be described by a subnanosecond correlation time,
reflecting the localized motion of the tryptophan, and a longer correlation
time corresponding to the motion of all, or a major portion of, the molecule.
While the values of the correlation times are smaller than those found by Ross
et al., a quantitative comparison is difficult in view of the different experi-
mental conditions. However, the magnitude of the shorter correlation time for
both sets of data is such as to suggest that it reflects the motion of several
amino acids.
It remains uncertain whether the rapid rotational mode reflects solely the
motion of the tryptophan with respect to the balance of the protein or
whether neighboring residues are involved. It also remains to be seen whether
only two rotational modes are strictly present or whether additional modes
may be detected with the development of techniques of higher resolution.
The amphipathic peptide melittin, which is isolated from bee venom,
consists of 26 amino acids.(48) The sole aromatic chromophore is a tryptophan
residue at position 19. In solution at low electrolyte concentration, melittin is
believed to exist as a largely structureless monomer. At high ionic strengths, self-
association occurs to form a tetrameric species, which is mostly
Lakowicz et al. have employed frequency-domain measurements to
examine the anisotropy decay of monomeric and tetrameric melittin. (49) In
each case, determinations were made in the absence and presence of the
quencher acrylamide. Dynamic quenching by acrylamide increases the fraction
of the total emission which occurs on the subnanosecond time scale, thereby
providing increased information about rotational motions in the picosecond
range. This is of particular usefulness in studying the internal motions of
proteins and peptides.
In 0.01 M Tris, pH 7, at 20 °C, where melittin is monomeric, the intensity
decay was found to be multiexponential and characterized by decay times of
0.2, 2, and 4 ns in the absence of quenching. The addition of increasing levels
of acrylamide resulted in a progressive reduction of the magnitude of the
decay times, which were equal to 20 ps, 260 ps, and 1.39 ns in 2 M acrylamide.
The anisotropy decay of monomeric melittin could be analyzed
acceptably in terms of two rotational modes (Figure 1.7). The corresponding
correlation times were essentially independent of the degree of quenching by
acrylamide, indicating that the decays of intensity and anisotropy were not
coupled and that acrylamide does not modify the rotational modes. Almost
60% of the anisotropy decay was associated with the more rapid rotational
mode, with a correlation time of about 160ps, while the remainder decayed
with a correlation time near 1.7 ns.
In the presence of 2 M NaCl, in which case melittin is tetrameric, the
intensity decay was also multiexponential and required the assumption of
three decay times for acceptable fitting. The decay times decreased from 0.2,
2, and 5 ns in the absence of quencher to 0.04, 0.3, and 1.2 ns in 2 M
acrylamide.
As in the case of monomeric melittin, the anisotropy decay was
biexponential. In this case the dominant rotational mode, which accounted for
about two-thirds of the anisotropy decay, corresponded to a correlation time
near 3.5 ns, while the balance of the anisotropy decayed with a correlation
time close to 60 ps.
The longer correlation times observed for both monomeric and tetrameric
melittin are in the range expected for rotation of the entire molecules, to
which they can probably be attributed. The more rapid rotational mode must
arise from some form of internal rotation involving tryptophan. It is of interest
that the correlation time associated with the more rapid rotational mode is
longer for monomeric than for tetrameric melittin (160ps versus 60 ps),
presumably reflecting the differing contribution of segmental motion involving
tryptophan in the two cases. It is also worthy of mention that no convincing
evidence was found for the presence of a correlation time of magnitude 1–2 ps,
which would arise from the unhindered rotation of a single indole group.
Time-domain measurements of the anisotropy decay of melittin have been
made by Tran and Beddard.(50) Their findings agree qualitatively with those
of Lakowicz et al. in that short and long correlation times were observed; the
magnitude of the latter was in the range expected for rotation of the
entire molecule. The principal difference between the two studies was in the
magnitude of the shorter correlation time, for which Tran and Beddard found
a value of 600–700 ps, which is substantially larger than that reported by
Lakowicz et al. An anomalously low value of 0.14 was found for A0 for
excitation at 300 nm. It is possible that the time resolution of this study was
insufficient to recover the early portion of the anisotropy decay, resulting in
a low value of A0 and an elevated correlation time.(49)
1.5.3. Anisotropy Decay of a Tightly Bound Fluorophore: Lumazine Protein
Visser et al. have reported time-domain measurements of the anisotropy

decay of the lumazine protein from Photobacterium leiognathi.(39) This protein,
which has a molecular weight of 21,000, contains fluorescent 6, 7-dimethyl-
8-ribityllumazine as a noncovalently bound prosthetic group. The anisotropy
decay of the lumazine fluorophore was monitored for both the free state and
when combined with protein.
The free lumazine derivative has a quantum yield of 0.45 and a
fluorescence decay time of 9 ns. The expected rotational correlation time for
a molecule of this molecular weight is of the order of 100 ps, which is
comparable to the width of the laser pulse employed This system
thus provides a rigorous test of the ability of time-domain measurements to
recover very short correlation times. Simulated data indicated that nearly all
of the decay of anisotropy occurred within the time course of the excitation
pulse.
The measured correlation time for free lumazine derivative was 70–80 ps
(20°C). Essentially the same value was obtained for the unquenched
fluorophore and for the fluorophore in the presence of 0.24 M KI, which
reduced the decay time of fluorescence intensity to 590 ps. If the aromatic
portion of the molecule is approximated by an oblate ellipsoid with semiaxes
of 4 and 2 an average correlation time close to 40 ps may be computed for
the unhydrated molecule. Since the ribityl side chain, as well as bound water,
would be expected to increase the observed correlation time, the measured
value is in reasonable agreement with that expected. This study, which
probably approaches the practical limits of time-domain measurements of
anisotropy decay, underlines the importance of rigorous deconvolution from
the excitation profile, especially for short correlation times. In the present
case, simulated data indicated that, in the absence of deconvolution, the
observed time profile of anisotropy is severely distorted by the finite duration
of the excitation pulse.
The anisotropy decay of the lumazine-containing protein was also
examined. It could be adequately fitted in terms of a single correlation time
of 9 ns (19 °C). The addition of sufficient KI to reduce the decay time of
fluorescence intensity from 14 to 3 ns did not alter significantly the computed
correlation time, although the shape of the observed anisotropy decay curve
was considerably distorted. This provided a confirmation of the adequacy of
the deconvolution procedure.
The lumazine-containing protein has a molecular weight of 2.1 x 104. An
anhydrous particle of this molecular weight with spherical symmetry would
have a predicted correlation time of about 6 ns. It is probable that the
difference between the predicted and observed values may be attributed to the
combined effects of hydration and of deviation from a strictly spherical shape.
The lumazine-containing protein can associate with luciferase to form a
protein complex of total molecular weight 1.0 × 105. The quantum yield of the
lumazine fluorophore is not altered by the complex formation. The dissocia-
tion constant may be controlled by varying such parameters as ionic strength
and temperature. In view of the constancy of the quantum yield and the
observation that the anisotropy decay of the lumazine protein in both the free
and complexed states can be adequately described by a single correlation
time, the preexponential factors ai in the equation
should reflect to the fractions of lumazine protein in the two states.

The correlation times of free and completely complexed lumazine protein
were found to be 20 and 60 ns, respectively, at 2 °C. Anisotropy decay
measurements on mixtures thus provide an excellent test of the ability of
current techniques to resolve correlation times of a similar order of magnitude.
The results were not entirely reassuring. If the sum of a1 and a 2 was held
constant and equal to the (invariant) limiting anisotropy and the values of a1,
allowed to vary so as to minimize the recovered values of

were much too short, being equal to 7 and 33 ns, respectively.
However, if was fixed at 20 ns, the computed value of which corre-
sponded to a minimal was close to 60 ns, as predicted, although the range
of values corresponding to acceptable fits was very broad.
In the present case the ambiguities of fitting probably arise from several
factors. The contribution of the longer component is less than that of the
shorter, so that the former predominates in the noisy portion of the data at
long times. Also measurements extended only over about 55 ns, so that
depolarization is not complete.
It is clear from these careful studies that the precise resolution of correla-
tion times which are not separated by orders of magnitude is a difficult
problem, which has yet to be completely solved.
In a related investigation, Kulinski et al. have monitored the time decays
of fluorescence intensity and anisotropy for the trytophan residue of lumazine
protein in both the apo and holo forms of the protein.(51) In both cases the
time decay of intensity was highly nonexponential, requiring the assumption
of a minimum of three lifetimes for acceptable fitting. The recovered lifetimes
include a short (0.1–0.6 ns), a medium (1.1–3.6 ns), and a long (6.1–6.6 ns)
component. The presence of the lumazine derivative reduces the relative
contribution of the long decay time, resulting in a decreased average decay
time, presumably because of radiationless energy transfer to lumazine.
The time decay of fluorescence anisotropy at 20 °C (excitation at 300 nm,
emission at 337 nm) was biexponential, in contrast to the behavior of the
lumazine fluorophore. A short correlation time of 0.3–0.5 ns was observed,
corresponding to a localized motion of the tryptophan, plus a longer correla-
tion time of 5–6 ns, arising from the rotation of a major fraction of the
molecule. It is of interest that the latter value is significantly shorter than that
observed for the lumazine fluorophore. This might arise from either a different
orientation of the transition moments with respect to the molecular axes, the
presence of an internal rotation sensed by the tryptophan, or a combination
of both.
1.5.4. Anisotropy Decay of a Transfer RNA
The presence of a natural fluorophore, the Y base, in the anticodon loop

of several tRNA species permits dynamic fluorescence measurements to be
made directly upon the unmodified molecules. In an early pioneering study,
Beardsley et al. carried out time-domain measurements of the decay of
fluorescence intensity and anisotropy for yeast tRNA Phe . (52)
The physical properties of tRNA Phe are modified by Mg 2+ ligation,
which results in changes in circular dichroism and a major increase in
quantum yield of the Y base. Beardsley et al. found that the intensity decay
could be described by a single decay time of 4.3 ns, which increased to 6.3 ns
in the presence of 10mM Mg 2 + . However, the quality of the fits was poor,
as judged from the values, suggesting the presence of unresolved multi-
exponential decay.
Parallel time-domain measurements of anisotropy decay were analyzed in
terms of a single rotational mode. Correlation times of 9.2 and 9.8 ns were
found in the absence and presence of Mg 2 + , respectively. The predicted
correlation time for a rigid anhydrous spherical molecule of the same
molecular weight is about 15 ns. Since the effect of hydration, or any deviation
from spherical symmetry, would be to increase the average correlation time,
the above value is a lower limit. It was accordingly concluded that internal
rotational modes involving the Y base were present and that tRNA P h e pos-
sesses a significant degree of flexibility.
Wells and Lakowicz have recently reexamined this system by making
frequency-domain measurements of intensity and anisotropy decay.(53) With
the improved resolution of the current instrumentation, it is evident that
at least two decay times are required to fit the intensity decay (Table 1.3).
In both the absence and presence of Mg 2+ , the intensity decay could be
adequately described by a short decay time of about 2 ns and a longer time
near 6 ns. However, the relative contributions of the two depend on the Mg 2 +
level. In the absence of Mg2 + , the amplitudes associated with the two decay
times are approximately equal; the addition of Mg 2 + increases the relative
amplitude corresponding to the 6-ns decay time to over five times that for the
shorter decay time.
A plausible explanation for the above observations is that the Y base
exists in two microenvironments which result in different decay times. A Mg 2 + -
induced conformational change favors the microenvironment associated with
the 6-ns decay time.
Anisotropy decay studies also indicated a major influence of Mg 2 +
(Table 1.4). In both the absence and presence of Mg 2+ , the anisotropy decay
could be accounted for in terms of two rotational modes, corresponding to
correlation times near 18ns and 0.3–0.4 ns. The presence of Mg 2+ substan-
tially increased the relative contribution of the slower rotational mode, which
now dominated the anisotropy decay. It is probably that the longer correla-
tion time represents the global rotation of the molecule, while the shorter
arises from a localized motion of the fluorophore. An obious explanation is
that the anticodon loop, which contains the Y base, is somewhat flexible in
the absence of Mg2 + , with a significant degree of mobility of the bases.
The binding of Mg 2+ constrains the anticodon loop into a relatively rigid
conformation in which the mobility of the bases is substantially reduced.
1.6. Anisotropy Decay of Biopolymers Labeled with an Extrinsic

Fluorophore
1.6.1. Anisotropy Decay and Internal Flexibility of Myosin
The muscle protein myosin is roughly Y-shaped, with a rodlike stem and
two globular (S-l) units at the head. A requirement of currently popular
models for the process whereby a myosin “cross-bridge” is linked to the thin
actin filaments within a muscle fiber and causes a mechanical thrust is that a
flexible hinge point be present within the myosin molecule.(54) Mendelson
et al. have employed anisotropy decay to obtain direct evidence for such a
hinge point.(54)
There is present in each S-l unit a single highly reactive sulfhydryl group,
which is convenient for the selective attachment of iodoacetamide derivatives.
Mendelson et al. employed the iodoacetamide derivative 1,5-IAEDANS
[N-iodoacetyl-N 1-(5-sulfoamino-l-naphthyl)ethylenediamine], which reacts
with sulfhydryl groups and whose fluorescence properties resemble those of
dansyl goups. The fluorescent conjugates prepared and studied by Mendelson

et al. contained about two AEDANS groups per molecule; labeling was
confined to the S-l head units. The labeled S-l units were also separated by
papain digestion and subsequent chromatographic purification.
For conjugates of purified S-l the time decay of anisotropy could be
described by a single rotational correlation time near 220 ns. Using the
equation of Belford et al. in the form it assumes for ellipsoids of revolution,
a series of predicted curves for anisotropy decay were generated as a function
of axial ratio (Eqs. (1.34)-(1.36)) for an ellipsoid with the
molecular weight (1.15 × 105) of S-l and an assumed hydration of 0.2. A region
was thereby identified in space which was consistent, within
experimental error, with the observed anisotropy decay. A lower limit for 3.5,
was attained at very low values of Since this minimal value agrees
with independent estimates of from other experimental approaches,(55) it is
likely that the label is preferentially oriented with its transition moments
roughly parallel to the axis of symmetry of S-l approximated as an ellipsoid.
The anisotropy decay found for myosin itself was also monoexponential
with a correlation time of 400–450 ns. This value, which is about twice that
for free S-l, is considerably less than that predicted for a rigid molecule with
the molecular weight (5 × 105) and asymmetry of myosin. Inasmuch as the
correlation time of an aggregated form of myosin increased to 1800 ns without
any significant change in the decay time of fluorescence intensity (20 ns), it
was felt to be unlikely that the value for monomeric myosin arose from some
form of localized motion of the probe. The favored interpretation was that the
low correlation time reflected primarily an independent rotation of the S-l
units within myosin. (54)
This interpretation was reinforced by parallel studies with heavy
meromyosin (HMM), which is formed by the action of trypsin upon myosin.
This fragment, whose molecular weight is 3.4 × 105, consists of both globular
S-l units joined by a fraction of the rodlike myosin stem. While the molecular
weight of HMM is about three times that of S-l, the measured correlation
time (400ns) was less than twice that of the latter fragment.
Although the interpretation is not entirely clear-cut, there is a definite
indication of some type of independent rotation of the S-l units within the
myosin molecule. However, this rotation appears to be somewhat hindered, in
view of the elevated correlation time, which is twice that of the isolated S-l
fragment. It is therefore unlikely that the S-l units are joined to the myosin
by some form of highly flexible universal joint.
In a later study Mendelson and Cheung reexamined the question of
possible interaction of the two S-l units as a possible factor in the elevation
of the correlation time of labeled myosin.(56) It is possible to remove a single
S-l head protein by limited proteolysis with papain. The resultant single-
headed myosin, in which the remaining S-1 unit was labeled with I AEDANS,
was found to have a correlation time essentially equivalent to that of native

myosin. This observation tends to rule out mechanical interference between
the two S-1 units as the primary cause of the increased correlation time of
myosin relative to that of S-1. A more likely origin is a significant degree of
stiffness of the polypeptide hinge joining each S-1 unit to the stem. The overall
model which qualitatively accounts for these results represents the S-1 units as
tumbling independently about the partially flexible joints connecting them
with the balance of the myosin molecule.
These findings are very relevant to proposed models for muscle contrac-
tion and, in particular, to the function of the S-l head proteins as cross-bridges
to the thin actin filaments. The ability of the S-l units to undergo segmental
motion independently of the myosin stem would render plausible the transla-
tion of the myosin molecules along the actin filaments by an “arm-over-arm”
movement.
1.6.2. Anisotropy Decay of a Fibrous Protein: F-Actin
The contraction of muscle and the mechanical force which is thereby

generated arise from the cyclic interaction of the two proteins myosin and
actin, which is coupled with ATP hydrolysis. The actin-myosin system passes
through several distinct states in the course of the overall process. The
dependence of the conformation of F-actin upon complex formation and upon
the presence of the various modifiers involved in muscle contraction is a factor
of central importance in any comprehensive model of muscle contraction at
the molecular level. Purified F-actin, the bihelical polymeric form of G-actin,
exists in solution as very long fibers, whose average length is of the order of
microns. If these were rigid and devoid of internal flexibility, their predicted
rotational correlation times would be immeasurably long.
The fluorescence dynamics of F-actin have been examined by Wahl and
co-workers, who utilized 1, 5-AEDANS conjugates of F-actin.(57) In the
presence of 0.1 mM Ca2+ the time decay of fluorescence anisotropy could be
acceptably fitted in terms of two rotational modes according to Eq. (1.64),
with the following values of the parameters:
The addition of the myosin proteolytic fragments S-l or heavy

meromyosin (HMM) (which consist of the globular head protein and of both
S-l units plus a portion of the stem, respectively) to F-actin in the presence
of 0.1 mM Ca2+ resulted in a progressive increase in σ2 to a limiting value of
1100 ns in excess HMM. The decay time of fluorescence intensity varied only
slightly, remaining close to 19 ns for all compositions. In the presence of
10mM ATP, which corresponded to dissociating conditions, the correlation
time reverted to that of free F-actin, thereby ruling out an artifact arising from
the denaturation of F-actin.
In the presence of the anisotropy decay parameters of
F-actin were substantially altered from their values in A biexponential
fit yielded in this case a1 =0.04, a2 = 0.25, =5.8ns, and = 682ns. The
longer correlation time thus undergoes a major increase in the presence of
The addition of HMM in the presence of resulted in a biphasic
response of the magnitude of the longer correlation time, which passed
through a minimum at a mole ratio of HMM to actin of 0.02, followed by an
increase. The addition of S-l under these conditions produced a similar
biphasic pattern.
These findings suggest that some kind of segmental flexibility exists in
F-actin; the degree of flexibility is dependent upon conditions. The shorter
correlation time, presumably arises from some form of localized rotational
motion, while the longer time, corresponds to the concerted motion
of a larger unit, probably a set of actin monomers. The initial decrease in
resulting from complex formation with HMM or S-l perhaps arises from an
increase in the flexibility of the links joining actin monomers, while the
increase observed at higher levels of HMM in the presence of and at
all levels of HMM or S-l in the presence of may reflect a stiffening of
these contacts.
The molecular dynamics of F-actin have also been studied by other
physical techniques, with results which differ quantitatively from those
summarized above. Thus, correlation times of and have been
computed from quasi-elastic light scattering and from saturation transfer
electron spin resonance, respectively.(58,59) In both cases a substantial increase
in correlation time occurred upon complex formation with HMM. While the
reasons for the varying results obtained with the different techniques remain
obscure, it seems clear that different molecular motions are being sensed by
these methods.
1.6.3. Anisotropy Decay for Proteins Displaying Internal Rotation Involving a

Well-Defined Domain: The Immunoglobulins
The antibodies of the higher vertebrates may be grouped into

three classes: IgG, IgA, and IgM. The most common of these, IgG, which has
a molecular weight near is composed of two “heavy” chains of
molecular weight and two “light” chains of molecular weight
The chains are joined by disulfide bridges to form a molecule which
is roughly Y-shaped. The IgA molecules are of similar structure, while the
IgM class consists of polymers of these basic molecular units.
The immunoglobulins, which normally arise in response to exposure to
molecules (antigens) which are foreign to the circulation of the animal, have
the biological function of combining with these antigens so as to facilitate

their removal. Potential antigens include the proteins and carbohydrates
present on the surface of invading microorganisms. Antibodies may also arise
from exposure to small groups, or haptens, which are conjugated to
homologous plasma proteins, which are not themselves antigenic.
The IgG immunoglobulins are bivalent, with two equivalent antigen-
combining sites per molecule. In the case of multivalent antigens, which
include protein and cellular antigens, a three-dimensional network is built up,
leading ultimately to an insoluble precipitate.
Papain cleaves the IgG molecules to yield an Fc fragment consisting of
most of the two “heavy” chains and corresponding to the stem of the
Y-shaped molecule, plus two Fab fragments, each of which consists of a
“light” chain plus the remainder of a “heavy” chain. An intact antigen-
combining site is present in each Fab fragment. The Fab fragments represent
the arms of the Y.
The IgG molecule may be regarded as an example of a multidomain
protein structure. The susceptibility to papain cleavage of the region near the
junction of the Fab and Fc subunits suggests that randomly coiled sections of
the “heavy” chains may occur here. This in turn raises the possibility of a
flexible hinge point in this region, which would permit some degree of
independent motion of the structural subelements.
Early static anisotropy studies upon dansyl-conjugated immunoglobulin
provided some indication of the presence of internal rotations, but were
quantitatively somewhat discordant.(60) The problem was subsequently rein-
vestigated by Wahl, who performed time-domain measuremnts of anisotropy
decay for a dansyl conjugate of γ-globulin.(61) The degree of substitution was
held sufficiently low (less than one dansyl group per molecule) to render
unlikely any significant depolarization by radiationless energy transfer.
The convoluted curves of both d(t) and s(t) at pH 8 showed a time decay
which was clearly nonexponential. Since adequate mathematical procedures
for analyzing such a complex system were not available at that time, an
empirical curve-fitting procedure was adopted. It was assumed that the time
decays of S(t) and D(t) could each be described in terms of two decay
times. By trial-and-error curve fitting, values of the two sets of decay times
were found such that the corresponding convoluted forms of S(t) and D(t)
reproduced graphically the observed curves of s(t) and d(t). The ratio
D(t)/S(t) then yielded the deconvoluted anisotropy A(t) as a function of time.
This proved to be also nonexponential. A repetition of the graphical curve-
fitting procedure was done to reproduce A(t) by an expression of the form of
Eq. (1.64), with a1 =0.075, a 2 = 0.14, σ 1 = 7.7ns, and σ2 = 123 ns The value of
σ2 is in the range expected for the rotation of the entire γ -globulin molecule
and may probably be loosely attributed to this origin. That of σ 1 can only
arise from some form of internal rotation. As the sites of attachment of the
dansyl label, as well as the possible contribution of hindered rotation confined

to the probe itself, were unknown, it was not possible to draw definite
structural conclusions.
Yguerabide et al.(62) subsequently made time-domain measurements of
anisotropy decay for IgG antibodies directed against the fluorescent hapten
ε-dansyl-L-lysine. Complexes of hapten with both intact IgG and its Fab
fragment were studied. The binding of hapten was accompanied by a 25-fold
increase in the quantum yield of fluorescence, so that it was readily feasible to
examine the fluorescence properties of the complex alone. In contrast to the
earlier studies, the sites of attachment of the fluorescent label were known and
specific.
The time decays of and were determined for the complex of
ε-dansyl-L-lysine with IgG and its Fab fragment. From these data, curves of
s(t) and A(t) were constructed. The time decays of s(t) were found to be
equivalent for IgG and Fab. These were nonexponential, indicating the
presence of multiple decay times. This is not unexpected, in view of the known
molecular heterogeneity of antibodies.
To minimize complications arising from convolution effects, analysis of
A ( t ) was confined to times after complete decay of the excitation pulse. This
approach has the obvious disadvantage of minimizing or missing altogether
the contribution of any rotational modes of very short correlation time, as
might arise from a localized motion of the probe. Unlike the case of s(t),
the time profile of anisotropy was very different for Fab and intact IgG.
The time decay of anisotropy was exponential for Fab, corresponding to a
single rotational mode of correlation time 33 ns. For a prolate ellipsoidal
molecule with the molecular weight of Fab (5.0 × 104) and a hydration of
0.3 ml/g, this value would be consistent with an axial ratio of 2.5. There is
thus no evidence for any internal rotation sensed by the probe, although, for
the reasons stated above, a rapid component of the anisotropy decay might
have been missed.
In contrast to the behavior of Fab, the time decay of anisotropy for
intact IgG was nonexponential and could not be fit on the assumption of a
rigid ellipsoidal shape. A least-squares fit of the observed curve of A(t)
indicated that it could be accounted for by Eq. (1.64), with a 1 =0.14,
The value of 1, is substantially smaller
than the minimum value (47 ns) predicted for a rigid, unhydrated spherical
particle of the same molecular weight and is of similar magnitude to that
found for the Fab fragment. The longer time, is roughly consistent with
that expected for the rotation of a major fraction of IgG.
The above results are compatible with a model in which the Fab sub-
molecules rotate with respect to the balance of the molecule. However, some
uncertainty remains as to the possible presence of rapid rotational modes of
shorter correlation time, arising from segmental motion within the Fab units.
The independent rotation of the Fab units may be a significant factor in the
antibody function, facilitating the combination with antigen.
More recently, these studies have been continued by Lovejoy et al.,(63)
who utilized rabbit antibodies directed against the hapten pyrenebutyrate
(PBA). This fluorophore has a much longer average lifetime than dansyl;
the value for the free hapten is about 100 ns for an air-saturated solution and
somewhat larger for an O2-free solution. The binding of PBA by the anti-
body resulted in a significant red shift of the primary excitation maxima
(from 326.5, 341.5 nm to 330.5, 347 nm) and of the emission maxima (from
375, 395 nm to 376, 396 nm). The average binding constant was sufficiently
high to permit virtually quantitative binding of hapten.
The time profile of fluorescence intensity decay was generally hetero-
geneous, except in the case of antibody produced with very long (11-month)
immunization times, for which a single fluorescence decay time of 157 ns was
found. The time decay of fluorescence anisotropy, which was similar for two
different preparations, was fitted to Eq. (1.64) by a least-squares procedure.
The values obtained were
The magnitudes of the correlation times are similar to those reported for a
different hapten by Yguerabide et al. Inasmuch as the time decay for PBA
could be monitored over a sufficient time interval to make possible the
accurate determination of the longer correlation time, these observations
strengthen the conclusion that mobility of the Fab units is a general property
of IgG molecules.
Holowka and Cathou have made analogous fluorescence dynamics
studies on the macroglobulin (IgM) class of antibodies.(64) Immunoglobulins
of the IgM class generally occur in animal sera as disulfide-linked pentamers
of total molecular weight near 900,000. Each monomer unit is somewhat
similar in structure to an IgG molecule, containing two light (L) and two
heavy chains linked by disulfide bonds and noncovalent interactions. A
third unrelated (J) chain is also present and may be involved in the assembly
of IgM from its subunits. A total of ten antigen-combining sites occur on its
ten Fab units.
Holowka and Cathou prepared IgM antibodies directed against
from horse, pig, and shark antisera obtained by immunization with a
dansyllysine streptococcal conjugate, which favors the formation of IgM
antibodies in these species.(64) The time decay of fluorescence intensity of
complexes with purified IgM molecules varied with the
species. For horse and pig IgM, the dominant component had a decay time
near 24 ns, while a secondary component had a decay time of 8–12 ns. In the
case of shark IgM the pattern was reversed, with the major component having
a short (4 ns) decay time. However, the contribution of the long decay time
was sufficient in all three cases to permit the monitoring of anisotropy for
times up to 200 ns.
Both horse and pig IgM displayed nonexponential anisotropy decay

which required at least two correlation times for fitting by Eq. (1.64). In both
cases a very long correlation time was observed, which presumably
reflected the global motion of the IgM pentamer, plus a shorter (61–69 ns)
correlation time, which must arise from some form of internal rotation. In the
case of shark IgM a third rotational mode of quite short correlation time
was detected. Also, the intermediate correlation time was somewhat
longer (93 ns) than the short correlation time for the other two species.
In the case of IgM no correlation time was observed which was equiv-
alent to that found for isolated submolecules. By pepsin or papain digestion
the fragments, of molecular weight 56,000, were obtained; these are
analogues to the Fab fragments of IgG. In parallel to the latter case, their
anisotropy decay was monoexponential, corresponding to a single correlation
time of 32–36 ns. By limited pepsin digestion the species was
obtained, corresponding to the two arms of the Y, plus a short connecting
segment of the stem; its molecular weight was 105,000–120,000. Its anisotropy
decay was nonexponential and required the assumption of two rotational
modes for fitting according to Eq. (1.64). The corresponding correlation time
were 38 and 211ns. The latter value is consistent with some degree of
independent motion of the units within the species.
While it is clear that segmental mobility on the nanosecond time scale is
present in IgM, in contrast to the IgG case it is not possible to make a clear-
cut identification of a rotational mode with the motion of a well-defined
submolecule. This may possibly be attributed to the hindrance of the rotation
of the , units by the quaternary structure of IgM.
In a subsequent study, Siegel and Cathou examined the effects of thermal
treatment (30 min at 60 °C) upon horse IgM antibodies against
The thermally treated IgM, as well as its fragment,
showed qualitatively a more rapid time decay of anisotropy than the corre-
sponding native species. While the magnitudes of the correlation times were
almost unchanged, their relative amplitudes were altered; the contribution of
the shorter correlation time increased substantially. The implication is that the
mobility of the unit is increased by limited thermal unfolding.
1.6.4. Anisotropy Decay of Calmodulin Complexes with TNS
Calmodulin, a Ca 2+ -binding protein of wide occurrence in eucaryotic

systems, is known to combine with, and regulate the activities of, a large
number of enzymes.(66) The combination with the regulated enzyme is
generally Ca2+-dependent.(66) Calmodulin functions as an initial receptor for
biological signals involving a change in the level of free Ca 2+ . (67)
The three-dimensional structure of the form of calmodulin

has recently been described.(68) It is roughly dumbbell-shaped, consisting of
two globular lobes joined by an strand (residues 66–92). The N- and
C-terminal lobes each contain two sites. The molecule, whose
molecular weight is 16,700, is rather asymmetric, being about long, while
the two globular N- and C-terminal lobes each have dimensions of about
The crystallographic structural determination was carried out for

calmodulin which had been crystallized from an acid medium (50 mM
cacodylate, It is of interest to determine whether the
crystallographic structure persists under more physiological conditions, as
well as whether any internal rotational modes are present.
Steiner and Norris have made time-domain measurements of anisotropy
decay for complexes of calmodulin with 2-toluidinylnaphtha-
lene-6-sulfonate (2, 6-TNS) under varying conditions of pH, ionic strength,
and temperature.(69) Calmodulin contains two binding sites for TNS; the
N-terminal (1–77) and C-terminal (78–148) half-molecules each contain a
binding site. The interaction is Ca2+-dependent, with little or no binding
occurring for the apoprotein. The TNS fluorophore is almost nonfluorescent
in aqueous solution, but acquires an intense fluorescence when bound to
calmodulin. Its use had the advantage of minimizing the contribution of
rotation confined to the fluorescent label, in view of its probable contact with
several amino acid side chains on the protain surface.
Time-domain measurements were made using a nitrogen pulse lamp as
source. While the pulse half-width was substantially longer than
those of the laser sources employed in other recent studies, this was probably
not a source of serious error, in view of the relatively long fluorescence decay
times encountered. Deconvolution and fitting by a least-squares procedure

were carried out by the methods outlined in Section 1.3.1.
The time decay of fluorescence intensity was multiexponential for all
conditions examined. The assumption of two or three components, depending
upon conditions, was required to obtain satisfactory fits. In each case a longer
decay time (12–16ns) and a shorter one (6–8 ns) were observed; at pH 6.5,
but not at pH 5.0, a third component of decay time was detected
(Table 1.5).
The time decay of fluorescence anisotropy was multiexponential, requiring
the assumption of at least two rotational modes for acceptable fitting
(Table 1.6). When corrected to the standard conditions of H 2 O, 25 °C
by multiplying by the ratio according to Eq. (1.28), the
magnitudes of the two correlation times did not vary greatly with temperature
(Table 1.6). In each case a longer correlation time of 10–13 ns and a shorter
one of 1–3 ns were detected. However, the relative amplitudes of the two rota-
tional modes showed a significant dependence upon conditions. At pH 5.0,
12°C, the anisotropy decay is clearly dominated by the slower rotational
mode. An increase in pH to 6.5 or an increase in temperature augments the
relative amplutide of the more rapid rotational mode.
It is logical to associate the shorter correlation time with a localized
motion of the label, while the longer correlation time may reflect the global
motion of the molecule. According to this model, the localized motions sensed
by the label are largely suppressed at pH 5 and low temperatures, which
approximate the crystallization conditions, and become more important at
more alkaline pH and higher temperature.
It is of interest to compare the magnitude of the longer correlation time
with that predicted from the theory of Belford et al. if the actual shape of
calmodulin is approximated by a prolate ellipsoid of revolution.(21) Such an
ellipsoid with the same length and molecular volume as calmodulin would
have an axial ratio near 3. From Eqs. (1.24)–(1.26), the computed value
of for an assumed hydration of 0.2 ml/g is 13 ns. This is in the range of

the observed values of the longer correlation time at pH 5.0. These results are
thus consistent with the crystallographic structure, provided that one of the
transition moments is roughly parallel to the axis of symmetry of the equivalent
ellipsoid, so that the anisotropy decay is controlled by this rotational mode,
since A2 = A3 = 0 from Eqs. (1.36). Superimposed upon this overall rotation is
a localized motion of the probe, which is dependent upon conditions.
References
1. P. Wahl, in: Biochemical Fluorescence (R. F. Chen and H. Edelhoch, eds.), Vol. 1, p. 1,
Plenum, New York (1975).
2. R. F. Steiner, in: Excited Stales of Biopolymers (R. F. Steiner, ed.), p. 117, Plenum, New York
(1983).
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2
Fluorescence Quenching:
Theory and Applications
Maurice R. Eftink
2.1. Introduction
Solute fluorescence quenching reactions were first applied to biochemical

problems in the late 1960s and early 1970s,(1– 7) and since that time they have
been a very valuable research tool for studies with proteins, membranes, and
other macromolecular assemblies. Quenching reactions are easy to perform,
require only a small sample, usually are nondestructive, and can be applied to
almost any system that has an intrinsic or extrinsic fluorescence probe. The
most important characteristic, however, is the value of the information that
these reactions can provide. Solute quenching reactions, using quenchers
such as molecular oxygen, acrylamide, or iodide ion, provide information
about the location of fluorescent groups in a macromolecular structure.
A fluorophore that is located on the surface of a larger structure will be
relatively accessible to a solute quencher that is dissolved in the aqueous
phase. A fluorophore that is removed from the surface of a structure will be
quenched to a lesser degree by the quencher. Thus, the quenching reaction
can be used to probe topographical features of a macromolecular assembly
and to sense any structural changes that may be caused by varying conditions
or the addition of reagents. In addition, quenching reactions can, in some
situations, provide information about conformational fluctuations. In Sections
2.3 and 2.4 I will discuss several examples of the use of solute quenchers in
studies with proteins, membranes, and nucleic acids.
Solute fluorescence quenching reactions can also be used to selectively
alter the fluorescence properties of a sample in order to resolve contributions
or aid in the measurement of data. To elaborate on this point, consider the
different characteristics of fluorescence: the quantum yield, excitation and
Maurice R. Eftink • Department of Chemistry, University of Mississippi, University,

Mississippi 38677.
53
54 Maurice R. Eftink
emission spectral positions, anisotropy, the time dependence of the intensity

(fluorescence lifetime) and anisotropy decay, and the wavelength dependence
of these parameters. If only a single emitting center exists in a sample, then
the interpretation of these fluorescence properties may be straightforward.
If, however, ground state (i.e., a mixture of fluorophores) or excited state
heterogeneity (i.e., excited state reactions) exists, the interpretation of steady-
state, time-domain, or frequency-domain fluorescence data will be difficult.
Solute fluorescence quenching provides the experimenter with another
variable (i.e., another variable axis, in addition to time and wavelength) which
may enable the resolution of fluorescence contributions. Consider Figure 2.1.,
in which a comparison is made of the dependence of fluorescence intensity on
time and quencher concentration. For the time axis, the relative intensity, F,
will decay in an exponential manner (Eq. 2.1) with lifetime, For the
quencher concentration axis, the Stern–Volmer equation (Eq. 2.2) describes
the drop in the steady-state intensity F with quencher concentration, [Q],
where Ksv is the dynamic quenching constant.
These equations apply to a homogeneously emitting sample; if the fluo-

rescence is heterogeneous, the various contributions will lead to a more
complex variation of the intensity with both time and [Q]. Even for a simple
system, however, Figure 2.1 shows that the drop in intensity along the time
and [Q] axes will be similar. In practice, a more extensive set of data is
usually collected in time-domain measurements, and one will typically be
Fluorescence Quenching: Theory and Applications 55
limited by a [Q] range of 0 to ~ 0.5 M. On the other hand, with quenching

studies the experimenter can select different quenchers (i.e., ionic, neutral,
polar, etc.) in order to achieve resolution. Several examples of the ways in
which solute quenching can aid in the resolution of fluorescence properties
will be given in Section 2.5.
In this chapter, I will limit consideration to solute quenchers, such as
molecular oxygen, acrylamide, and iodide, which quench by coming into
contact (or very close approach) with the fluorophore. Other types of
fluorescence quenching, due to long-range energy transfer, induced conforma-
tional changes, and various intramolecular reactions, will not be discussed.
Some of these topics are discussed in other contributions to this volume.
Phosphorescence quencing will be discussed, since it is a similar process.
I also point out that this chapter is not intended to be a comprehensive
review. Very many researchers are using solute quenching reactions, and I
cannot hope to cite all the work that has been done. Various aspects of the
method have been treated in reviews (8-12), and here I will focus primarily
on recent developments and applications of the method in biochemistry,
2.2. Basic Concepts
2.2.1. The Stern–Volmer Equation
The inverse of Eq. (2.2) is the classic form of the Stern–Volmer equation,
a relationship which describes the effect of quencher on the steady-state
fluorescence of a sample(13–15):
The dynamic Stern–Volmer constant, is equal to the product of a

quenching rate constant, times the fluorescence lifetime in the absence
of quencher. If quenching occurs only by a dynamic mechanism, then the
ratio where is the lifetime in the presence of quencher, will also be
equal (Note that this is true only if is a monoexponential
decay time.)
The dynamic quenching rate constant, will be the product of the
quenching efficiency, times the diffusion-limited bimolecular rate constant
for collision, k (the efficiency will be defined below). The value of k can be
theoretically calculated by use of the Smoluchowski equation (here we neglect

the transient term):
where D and are the sum of the diffusion coefficients and molecular radii,
respectively, of the quencher and fluorophore, and N' is Avogadro’s number
divided by 1000. The diffusion coefficient for each species can be predicted by
the Stokes–Einstein equation:
where is the solvent viscosity, is Boltzmann’s constant, T is absolute

temperature, and R is the radius of the species. Thus, for an efficient
quencher, is expected to vary with However, slight deviations from
Stokes–Einstein behavior are commonly observed.(14) For typical values of R
values in the range of
are expected for efficient quenching.
In Figure 2.2 is shown a plot of versus for the acrylamide
quenching of indole in various solvents. A reasonably good Stokes–Einstein
pattern is seen. These data also demonstrate that acrylamide quenching of
indole is a very efficient process in solvents of different polarity. In water at
25°C, the for the acrylamide quenching of indole has repeatedly been found
to be in the range of which is approximately

equal to the theoretical calculated via Eqs. (2.5)
and
Quenching may also occur by a static process, that is, a process that does
not involve diffusion. Usually both dynamic and static quenching occur
together, and a modified form of the Stern–Volmer equation is then used (see
below). When both quenching processes occur, a plot of versus [Q] will
usually be upward curving. According to the simplest theory, the plot
should represent only the dynamic quenching component. In Figure 2.3A is
shown a plot of and for the quenching of the fluorescence of indole
by acrylamide.
When there is ground state heterogeneity (i.e., more than one fluorescent
species), and only dynamic quenching is kinetically important, the
Stern–Volmer equation is
where is the dynamic quenching constant for the ith species and
is the fractional contribution of the ith species to the total fluorescence in
the selected excitation and emission wavelength regions. If there are two
components I and the for one is times larger than that for the
second, the Stern–Volmer plot of versus [Q] will be downward curving.
By fitting Eq. (2.8) to the data, the values of and can be determined
(see Section 2.6.2). In Figure 2.3B are shown data for the acrylamide
quenching of a mixture of 3-methylindole and 2-methylindole
Data for three emission wavelengths are shown, for reasons
which will be presented in Section 2.6.1. For each wavelength the plots curve
downward very slightly at low quencher concentration.
2.2.2. Quenching Mechanisms and Efficiency
A general kinetic expression for a fluorescence quenching reaction in an

isotropic phase is as follows:
Scheme 1
where A* is an excited state of a fluorophore, ( A * . . . Q ) is an encounter

complex, and (A ...Q) is some resulting complex in which the excess energy
has been dissipated as heat, The rate constants and are diffusional
rate constants for the formation and breakdown of the encounter complex;
is the rate constant for the internal quenching process. The electronic
mechanism for the internal quenching process is thought to be different for
different quenchers. Molecular oxygen and paramagnetic species, for example,
are thought to quench aromatic fluorophores by an electron spin exchange
process (leading to rapid intersystem crossing and thus facilitating conversion
through the triplet manifold to the ground state). (15,18,19) Acrylamide, other
amides, and amines appear to quench via an electron transfer process, that is,
transfer of an electron from the excited singlet state to the quencher to form
a transient charge transfer complex [in this case (A ... Q) above may be
written as For other quenchers, the quencher may be
the electron donor, and the excited state may be the acceptor.(23) Quenchers
that possess halogens or other heavy atoms appear to quench by enhancing
intersystem crossing via a spin–orbital coupling mechanism. (24,25) Still other
quenchers may act by a resonance energy transfer mechanism, when spectral
overlap exists. Except for energy transfer, the quenching mechanisms appear
to involve close contact between the excited state and the quencher, and thus
the quenchers may be considered to be contact quenchers (see below). Orbital
overlap is thought to be necessary for quenching by oxygen. Electron transfer
quenching may show a very slight distance dependence of exp
where is a measure of the size of the molecular orbitals, r is the actual
separation distance, and is a constant that is near unity. (243)
It is difficult to experimentally determine the quenching mechanism for a
particular quencher. This is especially true for cases in which the value of
is very large compared to that of and that is, when the efficiency is
near unity. The quenching efficiency, for the reaction in Scheme 1 is equal
to
This expression for should apply for most situations; see Ref. 9 for further
discussion and alternate expressions for then
This means that every time an encounter complex forms, quenching
follows. When will be equal to [see Eq. 2.5)], the rate constant
for collision between the quencher and fluorophore. If ,
then and the encounter complexes may dissociate before quenching
occurs. For such inefficient cases, will be equal to which of course will
be less than When fluorescence quenching reactions are applied to bio-
chemical systems, it is desirable to employ an efficient quencher–fluorophore
pair, so that the interpretation of values will be more straightforward.
Molecular oxygen seems to be efficient for virtually all aromatic fluoro-
phores,(26) but acrylamide and iodide(22) are not efficient for all common
fluorophores. Inefficient systems need not be completely avoided, but more
caution must be exercised in interpreting data with these. For example, it is

advised that, for an inefficient system, one sould always compare studies on
the quenching of a biochemical system with studies on the quenching of the
cognate fluorophore alone.(9)
The fact that the value of for an efficient quenching reaction is so large
makes it difficult to study structure–reactivity relationships that ordinarily
would aid in revealing the quenching mechanism. For example, we have tried
to demonstrate that acrylamide is a change transfer quencher by studying how
varies with the ionization potential of a series of fluorophores. (22) Since the
value of for acrylamide is so large (we estimate it to be for
indole and other fluorophores), very little variation in is found for various
fluorophores. The related quencher, succinimide, is less efficient for most
fluorophores, and a crude relationship between and the ionization poten-
tials of the series of fluorophores was observed.(22) In studies with other
fluorophore–quencher systems, correlations have been found between
values and the ionization potentials, electron affinities, or reduction potentials
of the reactants.(28–30)
2.2.3. Diffusional Nature of Quenching
In addition to questions regarding the step, the nature of the step

has been considered by several groups. (13,14,16,30–33) The step is of course a
diffusional process, and there has been discussion regarding the inclusion of a
time-dependent (transient) term for this rate constant. For a system with
spherical symmetry (with respect to one of the reactants) and for the case that
a reaction between two molecules, A and B, occurs when one of the reactants
approaches to within an interaction distance, of the fluorophore,
Smoluchowski(34) derived the following expression for the bimolecular rate
constant:
This complete version of Eq. 2.6 includes the transient term in the square
brackets; the symbols are defined above. In principle, is the sum of the van
der Waals radii for the two reactants, but in reality it may be slightly
larger.(32) Sveshnik off (35) was the first to apply this rate expression to solute
fluorescence quenching reactions, and, in doing so, he introduced a proba-
bility factor (i.e., an efficiency term) to account for the possibility that only
a fraction of the collisions may be effective in quenching. Collins and
Kimball(36) modified this theory to include the possibility that not every
approach to results in quenching. Instead, Collins and Kimball used the
so-called “radiation boundary” assumption to derive an expression for k(t).

They assumed that the reaction rate is proportional to the probability that one
reacting species is at a distance of between and from the second
species, and they introduced the specific rate constant k (in units of cm/s) for
the reaction. The latter can be considered to be the effective rate constant
when the species are close (i.e., within from one another); the
parameter is essentially the same as our in Scheme 1, when units are
adjusted (i.e., is converted to units of by multiplying by
The resulting expression for k(t) is
where and This expression is more

complex than that of Smoluchowski (Eq. 2.10), but for ordinary values of
and Eq. (2.11) simplifies to a form that is similar to Eq.
Yguerabide et al.(37) and Nemzek and Ware(32) showed how the above
radiation boundary rate constant applies to fluorescence quenching reactions.
For either Eq. (2.10) or (2.11), the effective quenching rate constant, will
be time-dependent. The transient terms [i.e., the second part of Eq. (2.10)]
relate to the very rapid reaction between A* and Q molecules that happen to
be near one another when A* is excited. In other cases, newly created A* will
not have neighboring Q molecules, and the quenching rate constant will be
described by the steady-state rate of diffusion given by Eq. [2.6].
The time-dependent bimolecular rate constants (Eqs. 2.10 and 2.11) thus
predict that fluorescence decay measurements, in the presence of quencher,
may be nonexponential. From Eq. (2.10), the apparent decay time in the
presence of quencher will be
Due to the transient term, the decay time will be time-dependent. At times
immediately following an excitation pulse, the decay will be rapid, as a result
of the term. For typical values of and
the transient term will be larger in magnitude than the steady-state term
until At the transient term will be only 5 % of
the steady-state term, and the transient term can be neglected at times longer
than this. This transient term will be folded into the steady-state decay rate
and will often be difficult to observe. For smaller D values, or for fluorophores
with smaller the transient term will be of greater significance.
Several workers have experimentally demonstrated the existence of such
a transient term in fluorescence quenching reactions. Nemzek and Ware (32)
demonstrated a nonexponential decay in the quenching of 1,2-ben-

zanthracene in a viscous solvent. Van Resandt(38) did likewise for the iodide
quenching of N-acetyl-L-tryptophanamide in water, using a picosecond laser.
Recently, Lakowicz et al.(39) have used a phase fluorometer, operating over
the range 10–2000 MHz, to demonstrate transient effects in the quenching of
indole by acrylamide and iodide. In Figure 2.4A are shown their data for the
acrylamide quenching of indole. In the pesence of quencher the frequency-

domain data indicate a nonexponential decay. Fits of Eqs. (2.10) and (2.11)
are shown in Figure 2.4B. Lakowicz et al. have found that the radiation
boundary relationship (Eq. 2.11) yields a much better fit to the lifetime data
than does the Smoluchowski relationship (Eq. 2.10). In fact, Lakowicz and co-
workers have suggested that the radiation boundary model may even be an
inadequate model to describe the data. Nevertheless, their fits with Eqs. (2.10)
or (2.11) were achieved with reasonable values of for the quenchers and
indole ring. The fitted D values were somewhat larger than expected with
Eq. (2.11), but were reasonable for Eq. (2.10). Lakowicz and co-workers have
also recently extended this treatment to the analysis of lifetime fluorescence
quenching data with proteins, and they believe that in some cases the
transient term makes a significant contribution to the quenching rate.(41)
More on this will be presented in Section 2.3.8.
2.2.4. Static Quenching
The transient effects described above will lead to upward curvature in

Stern–Volmer plots from steady-state fluorescence data.(32,33) In fact, upward-
curving Stern–Volmer plots have been routinely observed for efficient
quenchers, and several explanations (summarized in Refs. 40 and 43) have
been given over the years. In addition to (i) the above transient term in the
rate constant, the upward curvature could be due to (ii) the formation of a
chemically distinct ground state, nonfluorescent complex, and/or (iii) the
probability that a quencher and chromophore happen to be adjacent (without
necessarily interacting) at the instant that the latter becomes excited. Complex
formation can be easily included in the Stern–Volmer equation as follows (12) :
where is the association constant for the one-to-one complex. (In fact,
there are some cases in which complex formation is the dominant quenching
process; see, for example, Ref. 244 and references therein.) For the probability
of the nonspecific occurrence of quencher–chromophore neighbors, one can
define an “active volume” element, V, surrounding the chromophore. If a
quencher (one or more molecules) exists within this volume at the instant that
the chromophore becomes excited, “static” quenching is assumed to occur
instantaneously. In this case, the modified Stern–Volmer equation is (40)
Note that Eqs. (2.13) and (2.14) are similar, since is simply the expan-
sion of exp(x) when x is small. Either modified Stern–Volmer equation has
been used; we prefer the former only when a one-to-one complex is believed
to form.
Yguerabide et al.(37) and Nemzek and Ware (32) have shown that the
transient effect discussed above leads to the following modified form of the
steady-state Stern–Volmer equation, which is similar in form to Eq. (2.14):
where
The factor embodies the quenching that is caused by the transient term.
The term will lead to a slight upward curvature in a steady-state
Stern–Vomer plot. Furthermore, Andre et al.(33) included a factor for true
static quenching to give the following complete form of the Stern–Volmer
equation:
The combined factor exp(V[Q])/Y corresponds to an upward curvature, and

Eq. (2.14) will usually be adequate to describe data; using Eq. (2.14), the
apparent V value will then include contributions from both transient and true
static quenching.
To illustrate how the transient effect can lead to apparent static quenching
in intensity data, I show in Figure 2.5 simulations in which the Smoluchowski
equation (Eq. 2.10) is used for Shown are simulations for two values of the
diffusion coefficient The calculations
were performed by numerically integrating, over 50-ps intervals, the following
equation:
For both D values, upward-curving intensity Stern–Volmer plots are

predicted, even though no true static term was included. The solid lines in
Figure 2.5 show fits of Eq. (2.14) to the simulated data. The good fits
demonstrate that Eq. (2.14) can describe the data, regardless of the cause of
the apparent static effect. I also show calculated lifetime Stern–Volmer plots,
for lifetime values that would be measured by the phase lag method. Note that
the lifetime plots are also predicted to curve upward, although to a lesser
degree than the intensity plot, due to the transient effect. The dashed line
gives the dynamic quenching component, as given by the steady-state
Smoluchowski equation (Eq. 2.6). The that is obtained by fitting

Eq. (2.14) turns out to be reasonably close to the steady-state dynamic
Modified forms of the Stern–Volmer equation have been derived by
others who have taken into account certain other aspects of the mutual
correlation of reacting pairs, particularly at high concentrations of the
reactants.(43–46) We find the article of Peak et al.(43) to be very lucid. These
modified Stern–Volmer equations all provide for an upward curvature at high
[Q], and, again, Eq. (2.14) can be considered to be an approximation in each
case.
If there is ground state heterogeneity in a system and if one considers an
apparent static quenching constant for each component, then Eq. (2.8) must
be expanded as follows:
2.2.5. Various Quenchers
A list and description of useful solute quenchers is given elsewhere

(Table III in Ref. 9). To this list we can add trifluoroacetamide, which was
reported to quench tryptophanyl fluorescence in proteins,(47) and thallium
ion, which has been used as a quencher of various extrinsic fluorescent probes
on proteins.(48,49) Thallium ion also is an efficient quencher of indole
fluorescence and may prove to be a useful cationic counterpart of iodide
ion.(9) However, the poor solubility of certain thallium salts must be con-
sidered (i.e., the chloride salt has very poor solubility in water), the tendency
for thallium to precipitate proteins may be a problem, and its toxicity must
be recognized. Xenon gas or nitric oxide gas may prove to be useful.(50)
For any quencher–fluorophore pair, the efficiency, of the quenching
reaction must be determined in a model system. If the efficiency is much less
than unity (100%), downward-curving Stern–Volmer plots and unusual tem-
perature, viscosity, and solvent dependencies may be observed.(9,22,27) For
example, we found succinimide to be only about 70% efficient in quenching
the fluorescence of indole in water. This degree of inefficiency is not large, but,
on varying the solvent, we found succinimide to be a very inefficient (~ 10%)
quencher of indole in aprotic solvents.(17) It is necessary to appreciate this sol-
vent dependency of the quenching efficiency. In a protein or membrane
system, the fluorophore may be in a nonaqueous microenvironment. If so, the
ability of an inefficient quencher, such as succinimide, to quench fluorescence
will depend not only on the accessibility of the fluorophore, but also on its
microenvironment. Interpretation of quenching data can then be difficult. On
the other hand, a strong solvent dependence may make the quencher more
selective in quenching surface fluorophores.
2.3. Quenching Studies with Proteins
2.3.1. Exposure of Fluorophores
A common use of solute quenchers is to determine the degree of exposure

of intrinsic and extrinsic fluorophores in biochemical assemblies. The most fre-
quently studied systems are globular proteins and their fluorescent tryptophan
(Trp) residues. This will be the primary focus of this section. Some mention
will be made of other fluorophores, and other types of biochemical assemblies
(e.g., membranes and nucleic acids) will be discussed in Section 2.4. The
exposure of fluorophores will be related to the magnitude of the quenching
rate constant, (for an efficient quencher–fluorophore system). The term
accessibility is related to the quenching constant, That is, the accessibility
of a fluorophore depends not only on its exposure but also on its
fluorescence lifetime since
The charged quencher iodide can be used to selectively quench surface
fluorophore residues (2) (charge effects are also important; see below). The
neutral and polar quencher acrylamide can usually quench internal residues to
a small degree and shows a very large range of values(51) (see Table 2.1).
Molecular oxygen is a very efficient quencher, and, due to its small size and
apolar nature, it may penetrate into globular protein structures more readily
than the other quenchers.(52) Acrylamide is also an efficient quencher of
tyrosine fluorescence in proteins,(53) but its quenching efficiency for extrinsic
fluorophores is sometimes lower. (22,27) In Table 2.1 are given the range of
values of that have been observed for the quenching, by iodide, acrylamide,
and oxygen, of the Trp fluorescence of several single-Trp-containing proteins.
I briefly note that certain energy transfer methods (54,55) can provide
similar topographical information about the positioning of fluorophores in
proteins and membranes, but these methods are beyond the scope of this
chapter.
2.3.2. Effect of the Macromolecule's Size
The maximum value that is observed for proteins having an exposed

Trp residue, or other fluorophore, is about for iodide and
acrylamide(51); for oxygen, larger values are observed, due to its larger
diffusion coefficient in water. (52) The maximum value of found for the
quenching of a fluorophore attached to a macromolecule is expected to be
lower than that for the quenching of the free fluorophore. This of course is
due largerly to the reduced translational diffusion coefficient of the fluorophore
when attached to the macromolecule. The rotational mobility of the macro-
molecule also must be considered. Johnson and Yguerabide(68) have extended

a general treatment of Shoup et al.(69) to predict the dependence of a quenching
rate constant on the size of a macromolecule. Shown in Figure 2.6 is a model
for a quenching reaction between a fluorophore that is attached to a spherical
macromolecule, of radius and a uniformly reactive, spherical quencher, of
radius The cone angle describes the portion of the surface area on the
macromolecule that is occupied by the fluorophore. The full equation for the
quenching rate constant for such a model will not be presented here (see Eq. 6
of Ref. 68), but it is a function of the translational diffusion
coefficients, and and the rotational diffusion coefficient of the macro-
molecule, In Figure 2.7A is shown the predicted quenching rate constant
for the macromolecule-associated fluorophore divided by the rate
constant for quenching of the free (unattached) fluorophore as a
function of the size (in molecular weight units) of the macromolecule. As can
be seen, begins at 1.0 for an infinitely small macromolecule and
quickly drops to a plateau value of about 0.4–0.5 for a large macromolecule.
Thus, a fully exposed fluorophore attached to a macromolecule is expected to
show a quenching rate constant that is about 50% of the value for the free
fluorophore. This reduction in the maximum for a macromolecule-
associated fluorophore is important with regard to the proper evaluation of
experimental values. Only if the observed is less than ~50% of that for
the free fluorophore should one consider any additional shielding to exist.
Johnson and Yguerabide(68) further predicted the dependence of on
the fraction of the fluorophore’s surface area that is exposed on the
surface of the macromolecule. This dependence is shown in Figure 2.7B. It is
interesting that the dependence of on is not linear. At very low
degrees of true exposure (i.e., low a steeper slope is predicted.
2.3.3. Electrostatic Effects
In addition to the above constraints due to the size of the macro-

molecule, charged quenchers may experience additional limitations (or
enhancements) due to electrostatic effects. This has not been exploited very
often in quenching reactions, other than in a very qualitative manner, and
usually researchers attempt to minimize electrostatic effects by working at
high and fixed ionic strength. Comparison of the quenching by charged (i.e.,
and and neutral (i.e., acrylamide) quenchers can reveal the sign of
an electrostatic potential near a fluorophore attached to a macromolecule.
Ando and Asai (48) showed that by varying ionic strength one can, in principle,
determine the number of adjacent charges and their distance from the fluoro-
phore. The apparent quenching rate constant, for a charged quencher, will be
where is the rate constant in the absence of electrostatic effects, and

are the charge on the quencher and macromolecule (near the fluorophore),
respectively, is the elementary charge, D is the dielectric constant, is
distance of closest approach of the quencher and fluorophore, C is a constant,
equal to 1.02 for aqueous solution at 25°C, and is ionic strength. Thus, by
determining for a quencher of charge as a function of ionic strength,

one can in principle determine the charge near the fluorophore.
Ando, Asai, and co-workers(48,49) have applied this strategy to determine
the electric potential near two extrinsic fluorescence probes on heavy
meromyosin. The probes were covalently attached to two specific sulfhydryl
groups on the protein, and acrylamide, iodide, and thallium were used as a
neutral, on anionic, and a cationic quencher, respectively. In a similar manner
this group also characterized the electric potential around ATP bound to
this protein.(70)
2.3.4. Tryptophan Residues in Proteins
Among the single-Trp-containing proteins listed in Table 2.1, the ones

with the least exposed Trp residues, to each type of quencher, are apoazurin
from Pseudomonas aeruginosa (Pae) (Refs. 71, 72, and 77), asparaginase from
Escherichia coli (Ref. 71), ribonuclease T, from Aspergillus oryzae (Refs. 51,
56, 61, 71, 73, 74, and 78), and cod or whiting parvalbumin (Ref. 71, 75, 76,
and 237). Among proteins having two or more Trp residues, Trp-314 of horse
liver alcohol dehydrogenase (Refs. 56, 57, and 79–84), Trp-48 of apoazurin
from Alcaligenes denitrificans (Ade) (Refs. 59 and 77), and Trp-109 of
alkaline phosphatase from E. coli (Ref. 77) are found to be among the least
exposed to quenchers. For each of these Trp residues, little or no quenching
by iodide can be observed. James et al.(6l) reported a very small of
for the iodide quenching of ribonuclease at pH 5.5.
Very small values of are found for the acrylamide
quenching of the Trp residues in these proteins. For asparaginase, the
acrylamide is estimated to be less than or equal to
The oxygen quenching values for these proteins range from
to When compared to values for other proteins, these
oxygen are small. The value of for asparaginase is the
lowest that has been reported. Due to its small size, it is generally accepted
that oxygen can diffuse through proteins to quench internal Trp residues.
For the above-mentioned four proteins (and also Trp-314 of alcohol
dehydrogenase), the Trp residues apparently are buried, and some degree of
resistance to oxygen diffusion is afforded by the surrounding protein matrix.
The Trp emission is quite blue for asparaginase, apoazurin, ribonuclease
and parvalbumin. The ranges from about 308 (apoazurin) to 322 nm
and thus there is independent evidence to suggest that the
Trp residues of these proteins are in an apolar microenvironment. X-ray crys-
tallographic data for Pae azurin, (85) ribonuclease carp parvalbumin (87)
(a homologous protein, in which a Phe is substituted for the single Trp in the
cod protein), and alcohol dehydrogenase(88) are available and show the Trp
residues of these proteins to be buried with in the globular structures. Earlier

we noted that there is a crude relationship between the acrylamide quenching
for a protein and the for its Trp emission.(51) In Figure 2.8 we present
an updated version of this plot, which includes several new single-Trp
proteins. While some outliers exist (e.g., the for HSA is lower than expected
from its red , the general correlation of the acrylamide with
holds. Thus, the magnitude of can be considered a measure of the dynamic
exposure of these Trp residues.
The for oxygen quenching also seem to correlate with Trp exposure
although the range of is smaller. This is best seen from Figure 2.9, where
we plot the log for oxygen quenching versus the log for acrylamide
quenching for various single-Trp proteins. A good correlation is seen, with a
slope of 0.44. When oxygen quenching was first applied to proteins, it was
suggested that all Trp residues in proteins could be quenched by oxygen with
a very narrow range of That is, it was held that oxygen is not very
specific in its quenching ability. Figure 2.9 leads to a different conclusion. The
good correlation indicates that oxygen can be somewhat selective in sensing
the exposure of Trp residues in proteins. Figure 2.9 also contains several
points for the iodide quenching of single-Trp proteins. The slope is larger
(~1.6), and the plot indicates, as expected, that iodide is a more selective
quencher of surface Trp residues than is acrylamide. (Of course, electrostatic
effects play a role in the selectivity of iodide quenching.)
Static quenching is sometimes seen for the quenching of these single-Trp
proteins. This is most often seen for acrylamide as quencher, but some
examples with oxygen have also been reported.(57) Figure 2.10 shows data for
the oxygen quenching of asparaginase and ribonuclease These plots
show a comparison of and Stern–Volmer plots. The larger slope (and
upward curvature that is sometimes discernible) in the former is indicative of
a contribution from static quenching. Generally, such static contributions are
found to be smaller than the dynamic contribution. Human serum albumin
has an exceptionally large degree of static quenching by acrylamide (51) and
oxygen,(52) and the static component for oxygen quenching of asparaginase
(Figure 2.10), while small in magnitude, is relatively large when compared to
the dynamic quenching component.
In simplest terms, such static components must mean that there is a finite
probability that the quencher exists near a Trp residue at the instance of
excitation. One must keep in mind that the static quenching constants seen in
proteins are generally smaller in magnitude than those seen for quenching of
indole or tryptophan in water. Thus, the static quenching for proteins
apparently does not represent a large partitioning of quenchers into the
protein matrix next to the Trp residues, but it also indicates that quenchers
are often not excluded from a steady-state existence near Trp residues inside
proteins. In a following section, I will comment on transient quenching effects
in proteins. This phenomenon may also contribute to an apparent static
quenching, particularly when one compares intensity data and average
lifetime data that are measured at a single frequency (via phase fluorometry).
2.3.5. Ligand Binding and Conformational Changes
Knowledge of the value for the solute quenching of a particular

fluorophore in a biological structure is of interest, but quenching experiments
are especially useful for enabling the study of changes in the conformation of
macromolecules that may be induced by ligand binding or by changing pH,
degree of aggregation, etc. When there are several fluorophores, then the
interpretation of a change in the quenchability is difficult; one is usually
limited to concluding that there is a general increase or decrease in the
accessibility of the several fluorophores. Changes in the relative quantum yield
and lifetime of various fluorophores complicate any interpretation. If there is
only a single fluorescent group, interpretation of changes in quenchability is
more straightforward, particularly if fluorescence lifetimes are also available.
In Table 2.2 are given several examples of recent applications of solute
quenching to study changes in the conformation of proteins. The examples
range from small single-Trp proteins, such as melittin, ribonuclease
78 . Maurice R. Eftink
azurin, and phospholipase and the interaction of these with small ligands,
to very large lipoprotein and nucleoprotein complexes.
An elegant application of solute quenching, and other fluorescence
methods, is the study by O’Neil et al.(91) of complexes between calmodulin
and a series of basic, amphiphilic, peptides. These peptides contained
a single Trp residue, which was systematically positioned throughout the
sequence. In the resulting complexes, the accessibility of the Trp residues to
acrylamide was found to vary in a periodic manner (repeat unit of 3 to 4
residues), consistent with the periodicity of the
2.3.6. Mechanism of Quenching in Proteins—Penetration versus Unfolding

Mechanisms
To enable collisional quenching of internal fluorophores in proteins, it is

accepted by many, but not all, workers that some type of conformational
fluctuations in the proteins must occur. Since oxygen is so small, it is easy
to imagine that it will be able to penetrate into the crevices in a protein’s
structure and that only small structural fluctuations would be necessary for
oxygen to diffuse throughout a protein. (52,84) For larger quenchers, such
as acrylamide, there has been some question regarding the mechanism of
quenching of internal fluorophores. We suggested that, like oxygen, acryl-
amide can penetrate into the matrix of a globular protein, with such inward
diffusion being facilitated by rapid, small-amplitude fluctuations in the
protein’s structure. (73,110) Some have argued that, instead of the quencher
diffusing inward, a segmental unfolding of the protein occurs to increase
the exposure of the fluorophore to the aqueous phase and hence to the
quencher. (111–113) Others have raised the possibility that some types of
quenching occur over a distance, so that physical contact may not be required
for quenching internal fluorophores.(74) Also, the extent to which quenchers,
such as acrylamide and oxygen, are associated with proteins, and the effect
that such association would have on the interpretation of quenching rate
contants, has been discussed.(114) Here I will comment on these various
alternative interpretations.
The rate constant for an electron transfer reaction, is believed to
depend on distance as , where r is the center-to-center separa-
tion distance between the donor and acceptor, and is the contact distance.
If 7 is the contact distance for a typical quencher–fluorophore pair, then at
beyond van der Waals contact), the value of would be
only 5 % of that at This would correspond to a fluorophore that is com-
pletely shielded by an impenetrable layer of methylene groups. If quenching at
a distance of a few angstroms were to occur to a significant degree, one would
expect the value of calculated from experimental values via Eq. (2.6),
to be larger than the van der Waals In fact, for efficient quenchers such
as acryfamide, an of about 7 is calculated for the quenching of indole in
water. (40) This is close to the sum of the molecular radii of indole
and acrylamide Also, analysis of multifrequency phase/modulation
lifetime data for the indole–acrylamide reaction, in terms of the radiation
boundary form of the time-dependent Smoluchowski equation, yields
reasonable values.(39) Of course, the calculated value may be com-
promised by a slight degree of inefficiency, but the point is that model system
studies are consistent with requirement of contact for the quenching reaction.
Recently, we have prepared covalent adducts containing an indole ring
and an acrylamide moiety, which are separated by one or two bonds. We
find that intramolecular quenching occurs. Further study is needed to
evaluate the extent to which this represents quenching over a distance, or
quenching by intramolecular collisions between the groups.
At this time we cannot eliminate the possibility that some electron transfer
over a distance occurs in the acrylamide quenching of Trp fluorescence in
proteins, but it seems likely that quenching must involve very close approach,
if not contact, between the reactants.
The question of an unfolding process versus an inward penetration
process can be expressed by the two following kinetic schemes(84,117,123).
Here M and A represent a macromolecule and an attached fluorescence

probe, is the rate constant for diffusion of the quencher through the
solution to approach the surface of the protein (or to quench a surface
fluorophore; in the latter case the discussion in Section 2.3.2 applies), is
the rate constant for diffusion away from the surface, is the rate constant for
diffusion (penetration) of the quencher through the protein matrix to quench
an internal residue, is the rate constant for segmental unfolding of the
macromolecule, M, to form an altered conformation, M', and is the rate
constant for the reverse process. Figure 2.11 gives a visual depiction of these
two extreme kinetic models. In the penetration model, the final quenching
(e.g., electron exchange) step occurs within the protein matrix. Small-
amplitude fluctuations in the protein structure must facilitate the penetration
of quencher.(110,115–117,120) In the unfolding model, the final quenching step
occurs in the aqueous environment, at the surface of the protein. The symbols
for the penetration model are taken from Gratton et al.,(117) who provided a
thorough kinetic description or this model. The apparent rate constants for
solute quenching will be, for the penetration model,
Thus, expressions for the rate constant are of slightly different form. (These
models are analogous to the unfolding and penetration kinetic models for
hydrogen exchange in proteins.) (112,118) If the term in the denominator
of Eq. (2.20) is larger than then the two rate expressions are dis-
tinguishable, for Eq. (2.20) predicts a downward-curving Stern–Volmer plot
(for a single type of fluorophore!). If, however, in Eq. (2.20), then
the two rate expressions are not easily distinguished. In Eq. (2.19), may be
limited either by diffusion of the quencher through the solvent (when
or by penetration through the protein matrix (when In Eq. (2.20),
will be the product of the rate constant for diffusion through the solvent times
the equilibrium constant for the segmental unfolding transition.
The two models represent extreme kinetic mechanisms (in one the
quencher goes in, in the other the fluorophore come out), and in reality the
difference may be subtle. We suspect that for some buried fluorophores a
penetration mechanism may be a better model, while for others an unfolding
mechanism may be required. For some of the proteins with single internal Trp
residues we have tried to determine the most appropriate model by varying

the following parameters: temperature, bulk viscosity, quencher type, and
hydrostatic pressure. The basis for these studies is that, for a deeply buried
Trp residue, the penetration model gives (when whereas the
unfolding model gives In the former case, variation of with
temperature, pressure, viscosity, etc., will reflect variations in the penetration
step, For the unfolding model, variations in with these conditions will
reflect variations in both diffusion through the solvent and the unfolding
transition
Temperature dependence studies with ribonuclease and cod
parvalbumin(75) give apparent activation energies of 6–9 kcal/mol for acryl-
amide quenching. Figure 2.12 shows a recent redetermination, via lifetime
measurements, of the Arrhenius plot for the acrylamide quenching of
ribonuclease These activation energies are larger than expected for
diffusion through the solvent and could reflect the thermal activation of small-
amplitude fluctuations needed for the step. For an unfolding mechanism,
one might expect Kun to have a larger temperature dependence, if the
unfolding is similar to the thermal denaturation of the protein. However,
if the segmental unfolding is less extensive in nature, it is reasonable that

the apparent activation energy would be less, possibly as small as the
6–9 kcal/mol that we observe for quenching. As shown in Figure 2.13,
pressure dependence studies (up to 2600 bar) show essentially no variation
in the for the acrylamide quenching of ribonuclease and cod par-
valbumin. (119) This is reasonable for a penetration mechanism, where the
conformational fluctuations may be small in amplitude, like the mobile
defects described by Lumry and Rosenberg(120). Based on other studies of
the pressure-induced unfolding of proteins, (121,122) one would expect to
increase with pressure. Thus, an increase in would have been observed if
the unfolding model were appropriate for these proteins.
The acrylamide for ribonuclease and parvalbumin is found to show
little variation with bulk viscosity, between 1 and 10 cP, but to then show a
greater drop as viscosity is increased from 10 to Figure 2.14
illustrates viscosity dependence studies for ribonuclease and parvalbumin,

as well as the model systems NATA (N-acetyltryptophanamide) and
glucagon. The results with ribonuclease and parvalbumin are easily inter-
preted in terms of the penetration model. The rate-limiting step is at low
viscosity, but becomes at high viscosity. This will occur because
decreases, in a Stokes–Einstein manner, with increasing viscosity, whereas
may be relatively independent of bulk viscosity. We have adapted the idea of
Frauenfelder and co-workers(126) and have employed the Kramers equation to
describe the viscosity dependence of the and steps:
where is the magnitude of the rate constant (either or ) at and

x, the exponent of the viscosity, is some value between 0 and 1.0. If
the rate constant varies inversely with (Stokes–Einstein behavior). If
the rate constant is independent of bulk viscosity. Table 2.3 shows parameters
for a fit of Eqs. (2.19) and (2.21) to the acrylamide quenching data in
Figure 2.14 for ribonuclease and parvalbumin. In fact, the step is found
to have little viscosity dependence. We interpreted this as indicating that the
small-amplitude fluctuations needed for penetration of the quencher are
coupled very weakly to the viscosity of the bulk.
These viscosity dependence data are not easily interpreted in terms of the
unfolding model. For this model should vary inversely with and, for the
plateau region of versus to be explained, one would have to rationalize
that has the opposite viscosity dependence (i.e., decrease with from
that of In view of our understanding of protein unfolding refolding
transitions, such a dependence of on is not reasonable.(124,125) Thus, the
viscosity dependence studies strongly favor a penetration mechanism for
acrylamide quenching, at least for the two proteins studied.
In studies with different quencher types, one can vary the charge, size,
and efficiency of the quencher. One should, of course, avoid comparing
“contact” quenchers (see Section 2.2.2 for qualification of the term “contact”)
quenchers, such as oxygen, acrylamide, and iodide, with those which probably
quench over a distance (i.e., via resonance energy transfer), such as nitrite and
methyl vinyl ketone.(111) Figure 2.9 shows a comparison, for several single-Trp
proteins, of the quenching rate constants for oxygen, acrylamide, and iodide.
For proteins with buried Trp residues, such as ribonuclease and par-
valbumin, the rate constants vary in the order > acrylamide > iodide. Here
again, the pattern is indicative of a penetration quenching process, with
oxygen being very effective (large ) and the iodide ion being very poor (small
) at penetrating into the globular structures. If an unfolding mechanism were
to hold for all quenchers except oxygen, one would expect to see a similar
for most quenchers. This is because the would be the same for all
quenchers. This clearly is not the case for most proteins.
In studies with the slightly inefficient quencher succinimide, we obtained
what may be the strongest evidence for a penetration model. (17) Succinimide
has a quenching efficiency of about 0.7 in aqueous solution and is about 20%
larger in diameter than acrylamide. In comparative studies with several single-
Trp proteins, the ratio of the apparent quenching constants for succinimide
and acrylamide, was found to range from ~ 0.1 to
~0.7 (see Figure 2.15). Proteins having relatively buried Trp residues were
found to have small values of That is, succinimide quenches these with
a smaller rate constant than does acrylamide. Proteins with relatively solvent-
exposed Trp residues, such as glucagon and adrenocorticotropin, were found
to have larger values. This wide range of values could be due to the
critical size dependence of the dynamic penetration of quencher through a
protein matrix. However, we also discovered another explanation, that being
the inherent dependence of succinimide quenching on the microenvironment
of the indole ring. Whereas acrylamide is found to be ~100% efficient at
quenching the fluorescence of indole in all solvent ( being dependent only
on the inverse of the solvent viscosity, as shown in Figure 2.2), we found that
succinimide is a relatively inefficient quencher in aprotic solvents. For example,
succinimide is 70% as efficient as acrylamide in water, but is only 15% as
efficient in dioxane, 10% as efficient in acetonitrile, and 1% as efficient in
dimethylformamide. Thus, a low for a Trp residue in a protein could be
due to the aprotic microenvironment in which the quenching takes place.
While there are two possible explanations (i.e., a critical size dependence or
an aprotic microenvironment dependence) for the small for buried Trp
residues in proteins, both explanations are consistent with the dynamic
penetration model for solute quenching reactions. That is, low for buried
Trp residues can be explained as being due to the fact that succinimide is
slightly larger than acrylamide and thus will not penetrate as well or as being
due to the fact that succinimide, upon penetrating to reach an internal Trp,
experiences an aprotic microenvironment and thus will not quench well. The
unfolding mechanism, on the other hand, offers no means of explaining the
wide range of since in this mechanism the quenching reaction is assumed
to occur when a Trp is periodically exposed to the aqueous environment and
thus no dependence on the size of the quencher is predicted.
2.3.7. Interaction of Quenchers with Proteins
A concern in the interpretation of solute quenching studies is whether or

not a quencher interacts with a protein in such a way that the local concentra-
tion of quencher is increased. If there is a high local quencher concentration,
then the apparent values (obtained with reference to the bulk quencher
concentration) will give an overestimate of the kinetic exposure of the
fluorophore. Blatt et al.(114) have recently emphasized this point, in com-
parison with quenching studies with micelles (see Section 2.4.1). Based on
measurements of the acrylamide quenching of proteins as a function of
protein concentration, they calculated partition coefficients in the range of 30
to 100 for the interaction of acrylamide with serum albumin, monellin, and
-lactoglobulin. We have investigated this matter using both fluorescence
lifetime and intensity measurements and find no significant dependence of the
acrylamide quenching of serum albumin and monellin on protein concentra-
tion. ( 1 2 7 ) Furthermore, equilibrium dialysis measurements show no significant
interaction of acrylamide with serum albumin. Thus, we do not find evidence
for a high local concentration of this quencher in the globular structure of
these proteins.
The evidence presented above for static quenching suggests that there is
a certain probability that a quencher molecule (particularly neutral quenchers
like acrylamide and oxygen) can exist adjacent to a Trp residue at the instant
that excitation occurs. The magnitude of static quenching constants for the
quenching of Trp residues in proteins is less than that for aqueous indole,
however, for all proteins studied. Thus, the small degree of static quenching
in proteins does not indicate a strong binding of the quencher to the Trp
residues. The observed static quenching may be more correctly attributed to
transient effects as discussed below.
There is some evidence that certain quenchers may interact specifically
with certain proteins. For example, acrylamide is an inhibitor of the enzymatic
activity of alcohol dehydrogenase, which is not surprising in view of its
structural similarity to another strong inhibitor, isobutyramide. (81) Acrylamide
is also a weak competitive inhibitor of chymotrypsin, (127) a weak activator of
trypsin, (127) and a weak competitive inhibitor of cytochrome P450C-21.(128) For
several other enzymes, there is little or no effect of acrylamide on their
activity. (51,104,110) Acrylamide will covalently react with lysine and cysteine
side chains at high pH, (129,130) but there is no indication that the adducts
produced will act as quenchers.
The specific interaction of the charged quencher iodide with serum
albumin is well known, (67,131) and nonspecific electrostatic interactions
between a quencher and a macromolecule-associated fluorophore must always
be considered (see Section 2.3.3).
It is reasonable to suspect that the nonpolar quencher oxygen will parti-
tion weakly into the oily core of proteins. However, the degree of static
quenching by oxygen is not unusually large for most proteins.(52,71) Jameson
et al.(132) for example, fitted their data for the oxygen quenching of the
porphyrin (iron-free) fluorescence of myoglobin and hemoglobin with modest
partition coefficients of 0.3 to 0.6. Trichloroethanol is another nonpolar
quencher which one would expect to show a tendency to interact with oily
regions in proteins. This interaction may occur with human serum albumin,
but, for most proteins, quenching by trichloroethanol (at low quencher
concentration) shows a pattern similar to that by acrylamide.(133) At high
(0.2 to 0.5 M) concentrations, trichloroethanol appears to induce a change in
the conformation of some proteins. This solvent-induced transition is thought
to be similar to that induced by 2-chloroethanol, which involves interaction
with the nonpolar side chains of the protein and the subsequent unfolding of
the globular structure.(135)
It is always necessary to consider the possibility that a quencher interacts
with the system being studied. However, we believe that the evidence indicates
that the commonly used neutral quenchers, oxygen and acrylamide, do not
partition into proteins to a significant degree.
In cases where specific quencher–protein binding occurs, it does not
necessarily follow that this leads to enhanced quenching. If the quencher inter-
acts near a Trp residue, this would probably produce quenching, but interac-
tion at a remote site may not cause a change in fluorescence. An example
in which quenching can be attributed to the specific binding of quencher
is cytochrome P450C-21.(128) The binding of acrylamide results in a static
quenching of the Trp fluorescence of this protein, and the association constant
and static quenching constant are both found to be about 10
2.3.8. Transient Effects
There are data that demonstrate that the transient term of the
Smoluchowski equation (Eq. 2.10) must be included to fit time- and fre-
quency-domain measurements of the solute quenching of indole and other
simple fluorophores in isotropic solution.(32,33,37,39) Lakowicz et al.(39) have
studied whether such transient effects can be observed in the solute quenching
of Trp fluorescence in proteins. One protein that they studied was nuclease
from Staphylococcus aureus.(41) This single-Trp protein shows a fluorescence
decay which is nearly a single exponential. When acrylamide or oxygen is
added, the fluorescence decay becomes more nonexponential. This is illustrated
(for oxygen) by the phase–modulation data in Figure 2.16A. Notice that
Lakowicz et al. were able to use a modulation frequency as high as 2000 MHz
(as in Figure 2.4A) due to their novel application of a microchannel plate
detection system.(134) Such high frequencies are necessary to enable the short
lifetime contributions to be revealed.
The phase–modulation data for acrylamide-quenched nuclease can be
fitted by a double-exponential decay law. However, Lakowicz et al.(41)
demonstrated that the data can also be fitted by a transient effects model,
using either the time-dependent Smoluchowski equation (Eq. 2.10) or the
“radiation boundary” form of the Smoluchowski equation (Eq. 2.11). In fact,
Lakowicz et al. found that the radiation boundary transient equation provides
a superior fit for the solute quenching of nuclease by both oxygen and
acrylamide. This is illustrated in Figure 2.16B by the lower and the more
uniform deviation plots for the fits of the radiation boundary model. The
fitting parameters obtained for this model were D, the effective diffusion coef-
ficient for the quencher–fluorophore reaction, and k, the intrinsic quenching
rate constant (which is in units of cm/s, but can be converted to units of

as explained in Section 2.2.3). Tables 2.4 and 2.5 give such fits for
the quenching of nuclease and other proteins by oxygen and acrylamide.
Parameters for both the Smoluchowski equation (called the model) and
radiation boundary model fits are given. Note that for both fits the interaction
radius, can be a fitted parameter or it can be fixed.
This work of Lakowicz et al. is provocative and should spur further
experimental and theoretical work. Some may question the application of
theories derived for isotropic, homogeneous systems to diffusional processes in

an asymmetrically structured protein. Some steric factors must be considered,
and, in cases where penetration by the quencher occurs, the two-step nature
of the process (see Scheme 2 and Ref. 117) should be included in the inter-
pretation. These studies may also stimulate molecular dynamics calculations
to simulate the movement of a quencher toward Trp residues in proteins.
The relationship between transient effects and static quenching of steady-
state fluorescence is illustrated in Figure 2.5. It may prove to be valuable to

simultaneously analyze both steady-state and time- (or frequency-) domain
data for such transient effects. The possible existence of multiple quenching
rate constants and multiple fluorescence lifetimes, discussed below, may
further complicate the analysis for transient effects.
2.3.9. Multiple Quenching Rate Constants and Fluorescence Lifetimes
Both the rate constant for solute quenching and the fluorescence life-
time of a fluorophore in a protein may not be discrete values. Instead
there may be multiple or even pseudo-continuous distributions of or
values. (137,138,141) Consider the model for a protein in Figure 2.17. Three routes
are shown for the penetration of a quencher to an internal fluorophore. If this
is a reasonable model, then the apparent will have contributions form the
three routes. Furthermore, if the different routes have different activation
energies, then one would expect to see a curved Arrhenius plot. We have
carefully studied the temperature dependence of the acrylamide quenching of
ribonuclease (Figure 2.12), and we find a linear Arrhenius plot from 10 to
45°C. This indicates that either (a) there is one dominant rate for the collision
of the quencher with Trp-59 of this protein, or (b) that all routes have about
the same thermal activation energy. Figure 2.17 gives a model with multiple
but one could also argue for multiple values by consideration of the
existence of multiple conformational states of a protein.
There has also been much discussion recently about the fact that the
fluorescence decay of individual Trp residues in proteins is not usually mono-
exponential (see Refs. 66, 74, 137, and 138-143). This again may be due to the
existence of multiple conformations of proteins and may be best described as
a pseudo-continuous distribution of decay times. (140,141) In Section 2.6.3 I will
discuss the consequences of such nonexponential lifetimes on Stern–Volmer
quenching plots.
2.4. Studies with Membranes and Nucleic Acids
Solute quenching reactions have been used quite often in studies with
micelles and membrane systems. Much less has been done with nucleic acids.
Here we will focus on the significant differences in the application of the
solute quenching method to such structures. With micelles and membranes,
quenching reactions are controlled by the extent to which the quencher enters
the hydrocarbon-like subphase. Polar or charged quenchers do not enter, to
a significant extent, into most lipid subphases. As demonstrated by the work
of Shinitzky and Rivnay (44) or Pownall and Smith, (145) charged quenchers,
such as N-methylpicolinium, iodide, or cesium ions, can be used to determine
the aqueous surface accessibility of fluorophores in lipid assemblies. (Many
similar applications of charged and polar quenchers to assess surface
accessibility are given as entries 1–17 in Table 2.6.) Nonpolar quenchers can
partition into the lipid subphases, as discussed in Section 2.4.1 below, and this
can lead to enhanced quenching. Upward-curving Stern–Volmer plots are
often seen when nonpolar quenchers are used with micelles and membranes,
and this is probably due to the transient term in the Smoluchowski equation,
as well as true static quenching. Since diffusion is often limited to two dimen-
sions, a different form of the Smoluchowski equation must be considered
(Section 2.4.2). Some quenchers have been made to include a quencher moiety
as part of a fatty acid or phospholipid molecule. These “quencher lipids”
provide advantage in assessing the location and lateral mobility of membrane-
associated fluorophores and the quencher itself (Section 2.4.3).
Below we will expand on these aspects of quenching reactions applied to
micelles and membranes.
2.4.1. Partitioning of Quenchers into Membranes/Micelles
Solute quenching studies in micelles and bilayer membranes are often

controlled by the degree to which small solute quenchers are partitioned into
the hydrocarbon-like subphases of these structures, as well as the diffusion
coefficient of the quencher within these subphases. Apolar quencher
molecules, such as oxygen, chloroform, dimethylaniline, and nitromethane,
are favorably partitioned into micelles and membrane, and this enhances their
quenching effectiveness. Relationships describing the partitioning of solute
quenchers into micelles or membranes and the resulting Stern–Volmer
quenching pattern have been presented by several groups.(146–150) If a
fluorophore is found only in a micelle (or membrane vesicle; in the following
we will use the term lipid phase) and if the solute quencher also associates
with and quenches in the lipid phase, then a modified Stern–Volmer Equation
(Eq. 2.22) applies, but the quencher concentration term will be that in the
lipid phase,
where is the bimolecular rate constant in the lipid phase, and V is a static
quenching constant. If one defines a partition coefficient as
where is the quencher concentration in the aqueous solvent phase, then,
from Eq. (2.23), which is a conservation of mass relationship, one can derive
an expression (Eq. 2.24) for
where is the total quencher concentration over both phases, and

and are the volumes of the lipid phase and the solvent and the total
volume, respectively. Substituting this expression for into Eq. (2.22),
one obtains the following general Stern–Volmer equation for a phase parti-
tioning system:
This equation contains three unknown parameters and

presumably can be determined, or one can consider the product
as an unknown). When the lipid concentration is low the
above equation greatly simplifies. However, if one collects quenching data as
a function of the ratio (i.e., as a function of micelle/membrane concen-
tration), these three unknown parameters can be obtained by simutaneous
nonlinear least-squares analysis of the data sets. Alternatively, Blatt et al.(149)
have described a graphical fitting procedure.
Blatt, Sawyer, and co-workers(149,150) have also considered the possibility
that the interaction of the quencher with the micelle/membrane subphase may
be better described as a saturable binding process. In this case the expression
for becomes
where is the association constant, and n is the number of saturable binding

sites. By substituting this expression for into Eq. (2.22) a Stern–Volmer
relationship (not shown) is obtained for this case of quencher binding. The
value will then be the rate constant for reaction of the bound quencher with
the fluorophore and may involve jumping of the quencher from its binding
site to strike the fluorophore. Blatt et al.(149) discussed the possibility that
bound quenchers may quench only by a static mechanism. Regardless, the
resulting Stern–Volmer equation will have a maximum of four unknown
parameters ( and ), and simultaneous nonlinear least-squares
analysis of data sets at different ratios may enable fits to be obtained.
Again, a graphical procedure is described by Blatt et al.(149) for determining
the fitting parameters. These researchers have also pointed out that both
partitioning and binding of the quencher may occur together in a system, and
they have provided some interesting simulations. It should also be pointed out
that Eq. (2.25), and the discussion that follows, assumes that quenching
occurs only by lipid-associated quencher. If some quenching occurs by
quencher from the aqueous phase, an extra term must be added to
Eq.(2.25).
The advantage of a complete analysis of quenching data as a function
of the ratio is that one can (a) obtain intrinsic rate constants for
quenching, and (b) determine the way in which the quencher associates with
the lipid phase. If one were to work at a single ratio, only an apparent
value could be obtained, and it would be a function of (or n
and ), and that is, it would be incorrect to interpret such (app)
in terms of the microviscosity of the lipid phase. By resolving and
parameters are obtained that can be related to the physical characteristics of
the quencher and the lipid phase.(151) When values are large, they may be
difficult to determine. Omann and Glaser(148) have developed a protocol,
in which excess nonfiuorescent membrane vesicles are added, to aid in the
determination of large values.
In Figure 2.18 are shown typical data for the quenching in a compart-
mentalized system. The fluorophore is 2-(9-anthroyloxy)palmitic acid, which

is incorporated into egg phosphatidylcholine (pc) vesicles. The quencher is
5-nitroxide stearate. Stern–Volmer plots at four different lipid concentrations
are shown. These data were fitted to a model in which there is both parti-
tioning and binding of the quencher, with both dynamic and static quenching
occurring for each type of associated quencher molecules.(149)
Lakowicz et al.(146) studied the quenching of carbazole-labeled phospho-
lipids in vesicles by chlorinated hydrocarbons and found that the process
occurs primarily by the partitioning of the quencher into the lipid phase.
The fluorescence quencher oxygen has often been used to quench lipid-
associated fluorophores. Oxygen is favorably partitioned into lipid phases,
and thus an equation such as Eq. (2.25) is needed to describe the data.
Mantulin et al.(154) have recently studied the oxygen quenching of the Trp
fluorescence of apolipoprotein A-I complexes with dimyristoylphosphatidyl-
choline. They concluded that quenching occurs primarily by oxygen molecules
that are partitioned into the lipid phase.
2.4.2. Two-Dimensional Diffusion in Membranes
The Smoluchowski equation (Eq. 2.10) describes diffusional processes in

three-dimensional space. For collisional quenching reactions within a bilayer
membrane, the movement of the quencher and the fluorophore may be
constrained. The bilayer faces may act as boundaries. Consider a bilayer
of thickness h. If the diameter of the quencher and fluorophore is small
compared to h, then the normal Smoluchowski equation is probably adequate.
If, however, the size of the quencher and fluorophore approaches the bilayer
thickness, h, then diffusion will occur primarily in the plane of the bilayer.
For this case, Owen(155) and Blackwell et al.(l56) have presented versions of
the Smoluchowski equation for two-dimensional diffusion. The equation is
complex and will not be given here. The important feature, as pointed out
by Blackwell et al., is that the transient term can be very significant, in
comparison with the “steady-state” rate term, for two-dimensional diffusion.
This can partially explain why Stern–Volmer plots for membrane quenching
systems often appear to curve upward. Also, the “steady-state” quenching rate
constant is given by
(2.27)
as compared to for three-dimensional diffusion.

Fato et al.(157) performed a careful study of the quenching of the
fluorescence of a membrane-associated fluorophore, 9-anthroyloxy stearic acid,
by membrane-associated quenchers, ubiquinone homologues, in phospholipid
vesicles and mitochondrial membranes. They first measured quenching plots

as a function of phospholipid volume, to correct for incomplete partitioning
of the quencher and to determine its partition coefficient (see Section 2.4.1
above). Knowing the concentration of the quencher in the lipid phase, they
then calculated quenching rate constants and, using Eqs. (2.27) and (2.26) the
diffusion coefficients according to the two-dimensional and three-dimensional
diffusion models. For this system there was essentially no difference between
the calculated diffusion coefficients for the two models.
2.4.3. Quencher Moieties Attached to Lipid Molecules
A group of lipid-like molecules to which quencher groups have

been covalently attached have been employed in studies with membranes.
Among the latter category are nitroxide-labeled fatty acids and phospho-
lipids(151,158–161,164,165) and brominated fatty acids(136,162) and long brominated
hydrocarbons.(163) These molecules participate in the bilayer arrangement of
membrane systems and have proved to be very useful probes, particularly
since the quenching group can be attached at various positions along the fatty
acid chain.
For the most part such lipid-like quenchers can be considered to be
completely incorporated into membrane systems, but Chatelier et al.(161)
demonstrated with the quencher n-DOXYL-stearic acid that partitioning into
a purple membrane system is not always complete. These workers found that
quenching studies at various lipid concentrations were necessary to enable the
quencher partition coefficient to be determined and to enable
corrected Stern–Volmer plots to be constructed.
The nitroxide-labeled stearic acid derivatives are commercially available
with the nitroxide moiety at carbons 5, 7, 12, and 16. These derivatives have
also been included into phospholipids,(159) and a nitroxide-labeled cholesterol
derivative is available.(151) Holloway and co-workers(136,162) have prepared
dibrominated phosphatidylcholines with the bromines at carbons 6 and 7, 9
and 10, 11 and 12, and 15 and 16. The efficiency of these various quenchers
is difficult to assess. However, they all seem to require contact with the
fluorophores in order to quench. Also, the fatty acid and phospholipid
derivatives seem to align in a bilayer like normal lipids. For these reasons,
studies with a series of quenchers can be used to locate the position of a
fluorescing group. For example, Markello et al.(162) used the series of
brominated phosphatidylcholines to determine that the fluorescent Trp
residue of cytochrome b5, in phospholipid vesicles, is located approximately
7 beneath the bilayer’s surface (see more on this below).
With spin-labeled or brominated phospholipids, quenching appears
to involve a combination of static and dynamic processes.(159,164,166) As an
alternative to the Stern–Volmer equation, the following relationship has been

found to be useful for analyzing quenching data with these quenchers(159):
Here is the minimum fluorescence when the sample is fully quenched,

is the mole fraction of the quencher lipid in the vesicle, and n is the number
of quencher lipid molecules that are close enough to the fluorophore to result
in quenching. If n is as large as 6, this indicates that the fluorophore can be
completely surrounded by six quencher lipid molecules when (Note
that in a hexagonal lattice, a given phospholipid molecule will have six
neighbors.) Smaller values of n indicate that there is less overlap between
the fluorophore and the lipid quencher. In Figure 2.19 is shown a plot of
data, according to the above equation, for the quenching by a spin-labeled
phospholipid of the fluorescence of diphenylhexatriene, gramicidin, tryptophan
octyl ester, and -ATPase embedded into phospholipid vesicles.(159)
Chattopadhyay and London(250) have demonstrated that, by the use of
two such lipid quenchers with the quenching moiety at different locations
along the alkyl chain, the penetration depth of the fluorophore can be deter-
mined. By assuming (i) a random distribution of fluorophores and quenching
moieties and (ii) a static quenching process, these workers presented the
following relationship for the degree of fluorescence quenching by a lipid
quencher of concentration C (in units of molecule per unit of membrane
surface area):
Here is the effective quenching encounter distance, and Z is the vertical

distance, within the bilayer, between the fluorophore and the quenching
moiety (the equation assumes that if not, no quenching is predicted).
By using two similar moieties, placed at different positions along a lipid chain,
a parallax method is possible to determine the penetration depth of the
fluorophore (assuming that the penetration depth of the two quenching
moieties is known). This penetration depth, given as the distance,
between the fluorophore and the center of the bilayer, can be determined
by comparison of the relative fluorescence, in the presence of an
equal concentration, C, of quencher lipids #1 and #2, via the following
relationship:
where is the vertical distance between the bilayer center and the more
shallow quencher, and is the vertical distance between the quenchers
on the two quencher lipid molecules. Using pairs of nitroxide-labeled
phospholipids, Chattopadhyay and London(250) employed this parallax
method to determine the penetration depth of various membrane-bound
fluorophores.
The above type of lipid quenchers are also useful because they can par-
ticipate in phase transitions and phase separations like other phospholipids.
Measurement of the quenching by these agents as a function of temperature
can reveal differences in the fluidity of the gel and liquid-crystalline states.(166)
Also, London and Feigenson(159) have shown that the relative affinity of other
types of phospholipids for proteins can be measured via their displacement of
such lipid quenchers from the boundary region about embedded proteins.
These workers used this method to study the interaction of phospholipids
with -ATPase from sarcoplasmic reticulum (159) also see Ref. 165 for an
earlier, less specific application).
Another interesting strategy is to selectively incorporate a quencher lipid
molecule into one monolayer of a vesicle and to then observe the degree of
quenching of a fluorophore (i.e., protein) that is incorporated into the
opposite monolayer.(167)
2.4.4. Membrane Transport and Surface Potential
Two other interesting applications of solute quenching reactions are in

studies of the transport of species across membranes and the determination of
the surface potential of membranes.
Moore and Raftery (181) performed an elegant transport study by using
as a quencher and transport species. has about the same ionic radius
as , and they found that the acetylcholine receptor, embedded in mem-
brane vesicles, will facilitate the uptake of into the vesicles. A fluorescent
probe (8-amino-l,3,6-naphthalenetrisulfonate) was loaded into the inner
aqueous volume of the vesicles, and the inward flux of was then
monitored by quenching of the fluorophore, following stopped-flow mixing.
The inward flux was described by the relationship
where is the final, equilibrium concentration of inside the vesicles,

and k is an apparent rate constant for the flux. Combining this kinetic
relationship with the basic Stern—Volmer equation, the following equation
was obtained for the time dependence of the fluorescence signal:
The value of k, determined by analysis with the above equation, was further
related to a transport number per channel per second, from knowledge of the
vesicle size and the number of receptors per vesicle.
The electrostatic potential on a membrane surface can be estimated using
ionic quenchers, as demonstrated by Winiski et al.(182) These workers used
and tempamine (4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl) as cationic
quenchers of the fluorescence of 2-(N-hexadecylamino)-naphtalene-6-sulfonate
incorporated into phospholipid vesicles (which were neutral or negatively
charged by inclusion of phosphatidylglycerols). The apparent for this
reaction will be
where . is the quenching constant when charge effects are absent (i.e.,
neutral vesicles), z is the valency of the quencher, F is Faraday's constant, and
is the electrostatic potential sensed by the quencher adjacent to the
fluorophore.
2.4.5. Nucleic Acids
Most nucleic acids do not have intrinsic fluorophores (at room tem-
perature), and very few solute quenching studies have been performed with this
class of biomolecules. The Y base of phenylalanine tRNA does fluoresce,(183)
and the quenching of this Y base by acrylamide has been studied.(246)
Intercalating dyes and drugs, such as ethidium bromide provide extrinsic

fluorescence probes. Since some of these are pharmacologically important,
knowledge of the details of their binding is of interest. Lakowicz and
Weber(26) found that ethidium bound to DNA is very well shielded from
quenching by oxygen. The in this case is only about
This is lower than that observed for oxygen fluorescence quenching in any
protein system, and this indicates that intercalation of the dye between DNA
base pairs provides much steric protection. Zinger and Geacintov(247) have
studied the quenching, by oxygen and acrylamide, of three classes of DNA-
binding chromophores: those that intercalate (proflavin), those that bind into
the minor groove (Hoechst 33258), and bulky polycyclic aromatic hydrocar-
bons that partially intercalate (coronene). Oxygen was found to preferentially
quench the more exposed, groove-binding chromophore. Acrylamide was
found to be a relatively poor probe for the accessibility of the bound chromo-
phores, due to its low efficiency of the quenching. Atherton and Beaumont(184)
have studied the quenching of intercalated ethidium by the metal ions
and A fluorescent group can be produced in DNA by reaction
with malondialdehyde. Fluorescent cross-links are formed, and Summerfield
and Tappel(185) have studied the quenching of these cross-links by iodide
and Montenany-Garestier et al.(248) have used iodide quenching to
show that the Trp residue of a model tetrapeptide is not intercalated in a
complex with dsDNA. In studies with tRNA, Ferguson and Yang (186) have
covalently attached various extrinsic fluorescent probes at different places on
They then used iodide and acrylamide quenching to determine the
accessibility of these probes in the tRNA and its complex with Met rRNA
synthetase. Other fluorescent tRNAs have been made by forming a bimane
(188)
derivative and by incorporating into a specific
position; iodide quenching studies of these derivatives have been performed.
Most other applications of solute quenching to systems containing
nucleic acids involve the fluorescence of a protein that interacts with a nucleic
acid. Examples of this type of application are given as the last four entries in
Table 2.2.
2.5. Uses to Resolve Other Fluorescence Properties
Another general use of solute quenching reactions is to enable the resolu-

tion of heterogeneous emission from systems. As mentioned in Section 2.1,
solute quenching reactions provide the experimenter with a means of con-
trolling the fluorescence intensity and lifetime of a sample. Below we will give
examples in which quenching reactions have been used to dissect contribu-
tions to the steady-state, time-domain, and frequency-domain fluorescence of
a sample and to modulate excited state reactions.
2.5.1. Resolution of Steady-State Spectra
When there is ground state heterogeneity in the fluorescence of a sample,

such as is expected for a protein that contains more than one Trp residue,
solute quenching can be used to resolve the fluorescence spectra of the com-
ponents. This is easily done for the case in which one class of fluorophores is
accessible and another class is completely inaccessible to the solute quencher.
Lehrer(1) demonstrated this strategy by using iodide to selectively quench
certain Trp residues in lysozyme. With the addition of quencher, the fluo-
rescence of lysozyme shifted to the blue, and at high quencher concentration
the remaining fluorescence was attributed to the inaccessible class of Trp
residues. By difference the spectrum of the accessible class can be determined.
In Figure 2.20 are shown the emission spectra of apoazurin from Alcaligenes
denitrificans in the absence and presence of 0.45 M KI obtained in our
laboratory. This protein has two Trp residues, one of which is deeply buried
and the other of which lies on the surface of the protein.(77) Quenching by KI
allows the emission of the two residues to be separated. One of the best
applications of this use of quenchers to resolve spectra is the iodide quenching
of horse liver alcohol dehydrogenase. This protein also possesses only two
types of Trp residues; one type (Trp-15) lies on the surface of this dimeric
protein, and the other type (Trp-314) is buried at the intersubunit interface.
Laws and Shore(80) and Abdallah et al.(79) have shown that iodide selectively
quenches Trp-15, allowing the emission spectrum of the two residues to be
resolved.
As mentioned in Sections 2.2.1 and 2.6.1, solute quenching can be used
to determine the relative contribution (in terms of fractional fluorescence
intensities) of components (two or, at most, three) at any choice of excitation
and emission wavelengths, provided that the components have different
accessibilities to quencher. Recently, we have compared the dissection of
component spectra by solute quenching with that obtained by phase-resolved
spectral measurements for various two-Trp proteins.(59)
2.5.2. Resolution of Fluorescence Lifetimes
The analysis of fluorescence decay (time or frequency domain) of a

heterogeneous sample can also enable the determination of the fractional
contribution from components. This, of course, is only possible if the fluo-
rescence lifetimes of the components are sufficiently different. By combining
fluorescence decay measurements with solute quenching, one can often
achieve resolution of the components and obtain both the lifetimes and
exposure of the components. In fact, in cases where the initial component
lifetimes are similar, the detection and resolution of heterogeneity may only be
possible by the addition of a selective solute quencher.
In studies with proteins, Ross et al.(l89) combined iodide quenching and
pulse-decay measurements to obtain the individual Stern–Volmer quenching
plots for the Trp residues of alcohol dehydrogenase. Demmer et al.(190) have
recently repeated this study and have found that one of the Trp residues
of alcohol dehydrogenase (Trp-15) decays in a nonexponential manner.
Torgerson(191) and Robbins et al.(192) have also employed acrylamide
quenching to obtain individual Stern–Volmer plots for the fluorescence
components of myosin S-l and terminal transferase. Wasylewski and Eftink (93)
have used phase fluorometry in a similar manner to obtain the values for
iodide quenching of the individual Trp residues of metalloprotease. Even for
the single-Trp protein, ribonuclease Chen et al.(74} have determined the
values for acrylamide quenching for the two lifetime components of the Trp
at pH 7, using the time-correlated single-photon counting technique. The
results of their study are shown in Figure 2.21.
Use of global analyses, to be discussed in Section 2.6.1 (see also Chapter 5

in this volume), should greatly enhance this type of combined measurement.
2.5.3. Resolution of Anisotropy Measurements
For a system with ground state heterogeneity, the steady-state anisotropy,

r, of the emission will be the weighted average of the component values:
If one can selectively quench the emission of one of the components, the
component values can be obtained. For example, by selectively quenching
Trp-15 of alcohol dehydrogenase with acrylamide, a limiting anisotropy of
about 0.265 is reached at high [Q] (at =300nm and 20°C). This can be
assigned to the anisotropy of the inaccessible Trp-314 residue. From the r
value at [Q] = 0 and the values, the anisotropy for Trp-15 (r = 0.210) can
also be calculated.(193)
For systems in which there is a single fluorophore, measurement of r as
a function of [Q] can be used to construct a Perrin plot. From the slope of
this plot one can determine the rotational correlation time, for the emitting
center. This is because a dynamic solute quencher will cause a lowering of the
fluorescence lifetime. The r value is dependent on the ratio as given by the
Perrin equation:
Here is the limiting fluorescence anisotropy of the fluorophore in the absence

of motion. Equation (2.35b) is a combination of the Perrin equation and the
Stern–Volmer equation, where we have substituted
Accordingly, a plot of 1/r, obtained as a function of [Q], versus
will have a slope/intercept ratio of If is known,
can be calculated. Thus, from steady-state quenching measurements one
can determine the rotational correlation time of a fluorophore. The term
may seem complicated, but it is just a factor proportional to
the fluorescence lifetime, when the fluorescence is homogeneous. If the static
quenching constant, V, is zero, then this term is just For this
reason, this method has been referred to as lifetime-resolved anisotropy
measurements, even though it is a steady-state method.(56,60)
In Figure 2,22 are shown plots of Eq. (2.35b) for the acrylamide
quenching of several single-Trp proteins. (60) Lakowicz and co-workers (56,58)
have used oxygen as a quencher of a large number of peptides and proteins.
In Figure 2.23 is an example of their use of oxygen quenching and anisotropy
measurements to study the monomer tetramer equilibrium in melittin.
The ^'-intercept in Figures 2.22 and 2.23 is The limiting
anisotropy, of the fluorescence of the fluorophore will usually depend on
excitation wavelength, when there is more than one absorption oscillator. For
tryptophan and indole in a low-temperature, vitrified solvent, for example,
the anisotropy shows a distinct dependence on and the reaches a
plateau at 300 nm due to selective absorption into the band.(194,195) The
value at 300 nm is about 0.31 ± 0.01 for immobilized indole.(194–196) If the
that is found from a modified Perrin plot (Figures 2.22 and 2.23)
is much less than the limiting value of this indicates the occurrence
of very rapid motion of the fluorophore that is independent of global rotation
of the macromolecule. One can reasonably assume that this rapid motion
will be limited within a cone.(56) The cone angle, can be calculated
as For the data in Figure 2.22, the N form of
human serum albumin and the -deficient form of parvalbumin are found
to have much smaller than the other forms of these respective
proteins. In Table 2.7 are summarized anisotropy data obtained for several
single-Trp proteins, by use of oxygen and acrylamide as solute quencher.
Solute quenching can also aid in the analysis of anisotropy decay
measurements. This has been demonstrated by Lakowicz et al.,(197) who
progressively quenched samples of the peptide melittin with acrylamide and
measured the resulting intensity and anisotropy decays (frequency-domain
measurements). The dynamic quencher reduces the mean lifetime of the single
Trp residue in melittin. By enabling fluorescence data to be collected at
shorter times (higher frequencies), the contribution to the anisotropy decay
from rapid, picosecond motion becomes enhanced. In the study with melittin,
in both its monomeric and tetrameric forms, Lakowicz et al. were able to
resolve 60- and 160-ps rotational correlation times, in addition to the longer
correlation times for global motion of the peptide, by quenching with up to

2 M acrylamide. The picosecond rotational correlation times certainly exist in
the absence of quencher, but the accurate determination of these small values
on unquenched samples is difficult. Lakowicz and co-workers (246,249) have
also used this quenching/differential phase method to aid in the evaluation
of the rotational correlation time of smaller molecules, such as indole and
Y base. This strategy of using solute quenchers to lower the mean decay
time can also be useful with pulsed single-photon counting anisotropy decay
measurements. (198)
2.5.4. Resolution of Energy Transfer Experiments
Resonance energy transfer, from a donor fluorophore to an acceptor

(which may or may not fluoresce), is an excited state reaction that can
compete with solute quenching reactions in certain systems. An experimenter
can take advantage of this competition to try to resolve energy transfer paths
between multiple donors and acceptors.
Consider the simple system in Figure 2.24 where there is a single acceptor
(A) and three donor ( and ) fluorophores (e.g., they may be Trp
residues). Let be so close to A that its transfer efficiency is >90%. Let
be located at a distance approximately equal to the distance for 50%
transfer efficiency. Let be located well beyond the distance so that its
transfer efficiency is <10%. Further, let A be fluorescent so that sensitized
emission can be observed, and let A not be directly quenched by a particular
solute quencher. If one were to excite exclusively into the donor fluorophores,
the following patterns would result when the solute quenching of sensitized A
emission is observed. Energy that is transferred from to A would not be
significantly affected by the quencher. Since transfers little energy to A,
solute quenching of would cause only a small, indirect quenching
of A. Solute quenching of would, however, lead to a significant quenching
of A. The point is that a solute quencher will most effectively quench the
sensitized emission of an acceptor by quenching those donors that are at a
distance of from the acceptor (not by quenching those donors that are
closest to the acceptor, as has been claimed by some).
The analysis of the time- (or frequency-) domain kinetics of resonance
energy transfer reactions can, in some instances, be aided by employing
solute quenching. To illustrate this usage, consider the frequency-domain
data in Figure 2.25 for the Trp-to-NADH energy transfer in an alcohol
dehydrogenase–NADH complex. There are two Trp residues that can transfer
energy to bound NADH. The surface Trp residue, Trp-15, can be selectively
quenched by iodide, leaving only Trp-314 as an energy donor. Figure 2.25
shows that the multifrequency phase and modulation data are altered by the
quenching of Trp-15. In another example of the use of solute quenching to aid
in the analysis of energy transfer reactions, Gryczynski et al.(218) have used
steady-state solute quenching to evaluate the distribution of end-to-end
distances of flexible molecules having a fluorescence donor and acceptor at
either end.
2.5.5. Other Uses of Solute Quenching
The competition between dynamic solute quenching and various excited

processes makes possible other practical uses of these reactions in addition to
the ones mentioned above. Solute quenching may compete with excited state
chemical reactions, and thus quenchers can sometimes be used to protect
chromophores from undergoing photolysis.(204) Of course, a quencher may
also potentiate photolysis if the quenching mechanism involves, for example,

irreversible electron transfer.(205) Solute quenchers can compete with triplet
state decay (see Section 2.7), and some quenchers (i.e., heavy atom quenchers)
can promote intersystem crossing and thus can lead to an increase in the
population of the triplet state.
Solute quenching reactions can compete with excimer formation. For
example, Chong and Thompson(206) used oxygen quenching to attenuate the
extent of excimer formation by pyrene-labeled sphingomyelin in phospholipid
vesicles. From steady-state spectra, as a function of relative oxygen concentra-
tion, and the fluorescence lifetime of the unquenched pyrene monomer, these
researchers were able to determine the apparent rate constant for excimer
formation. The latter rate constant provides information about the fluidity
of the bilayer.(207) This quenching method offers several advantages over the
standard method for determining the pyrene excimer formation rate constant.
The quenching method does not require the measurement of the lifetime or
intensity of the excimer and requires knowledge only of the relative quencher
concentration.
There can also be competition between solute quenching reactions where

two different types of quenchers are used. In fact, one of the early means of
determining that a quencher is a dynamic quencher was to determine the
apparent value for the quencher in question as a function of the concen-
tration of a second quencher that was known to be a dynamic quencher. If the
apparent for quencher no. 1 drops as quencher no. 2 is added, then the
first quencher must also be dynamic. Examples of the use of two quenchers in
studies with proteins can be seen with liver alcohol dehydrogenase. One can
quench most of the fluorescence of the surface Trp residue, Trp-15, of the
protein by adding iodide or acrylamide. Hagaman and Eftink (57) added
acrylamide to this protein to then enable quenching of the internal residue,
Trp-314, by oxygen to be studied. Similarly, Laws and Shore(80) quenched
Trp-15 with iodide to enable the alkaline quenching of Trp-314 to be
characterized. Somogyi et al.(208) have presented a general discussion of the
use of two quenchers (so-called double quenching) to further resolve the
heterogeneous emission of proteins. It is best to use a very selective quencher
(i.e., ionic quenchers such as iodide), which will strike primarily surface
fluorophores, in combination with a less selective quencher (i.e., oxygen or
perhaps acrylamide), which can quench internal fluorophores to a certain
degree. By performing quenching studies with one quencher as a function of
the concentration of the other, the accessibility of the various classes of
fluorophores (surface and internal) can be quantitated, in optimal cases.
Another practical use of solute quenchers is to produce a fluorescence
change, where one did not exist before, to enable thermodynamic or kinetic
studies to be performed. For example, consider a macromolecule–ligand
complex in which the fluorescence of the macromolecule is unchanged by the
binding of the ligand. In this case, fluorescence measurements will normally be
of little value in studying the binding of the ligand. However, suppose that the
accessibility of fluorophores on the macromolecule to solute quenchers is
altered by ligand binding. If so, the addition of the quenchers will enable
ligand binding titration curves (or kinetic binding data) to be obtained using
fluorescence spectroscopy. Messmer and Kagi(209) developed this argument
in their study with creatine kinase. The Trp fluorescence of this enzyme is
quenched by only 9–15% upon the binding of ATP. The Trp residues of
creatine kinase are partially accessible to iodide, but, when ATP is bound, the
accessibility drops to essentially zero. For such a system at intermediate
concentrations of the ligand, L, the following Stern–Volmer equation applies:
where is the dynamic Stern–Volmer constant for quencher, Q, for the

protein alone, is the ligand dissociation constant, n is the ratio of the
Stern-Volmer constant for the protein–ligand complex to that for the protein
alone and R is the ratio of the fluorescence intensity of the
complex to that of the protein alone. The apparent for this system
will be
If R = l.0 (i.e., the binding of ligand does not quench or enhance the
fluorescence of the protein) and at the ratio of the initial
in the presence of L to that in the absence is
and it can be seen that can be obtained from a plot of versus

[L]. Messmer and Kagi(209) obtained dissociation constants for the inter-
action of ATP, ADP, and AMP with creatine kinase in this manner, using
either iodide or acrylamide as the. solute quencher.
The reduction of the fluorescence lifetime of fluorophores by added
quenchers can also be used to establish lifetime reference standards.(210)
2.6. Recent Developments in Data Analysis
It is always risky to entitle a section “Recent Developments...,” especially

in the area of fluorescence spectroscopy, because one's comments can become
quickly outdated. Developments in picosecond flash and phase fluorometry
will be reviewed by other authors in this series. Here I will comment primarily
on developments in data analysis.
2.6.1. Simultaneous Analyses of Quenching Data
While others have certainly contributed,(211) Brand and his proteges(212–217)

have been foremost in promoting the strategy of overdetermining fitting
parameters by simultaneously analyzing multiple, linked data sets (see also
chapters in this volume). This has been applied primarily to time- and fre-
quency-domain fluorescence lifetime measurements,(212,213) but global analyses
can be performed for any cases in which data sets can be linked. For example,
if Stern–Volmer quenching data for a sample have been obtained at a set of
emission wavelengths, or at various temperatures, or pressures, or in the
presence of various ligand concentrations, etc., one could simultaneously fit
the data sets to obtain quenching constants plus other constants that depend
on the nature of the linkage (i.e., when emission wavelength is varied, the
other fitting parameters are the wavelength dependence of the fractional inten-
sities of the component spectra). Beechem and Grattan (215) have generalized
a global fitting routine that will apply to many types of fluorescence data,
including solute quenching. We believe that in the near future global analysis
of solute quenching data sets will become a standard procedure for extracting
quenching constants and other fitting parameters. This will particularly be so
with the expanding use of on-line data acquisition and other equipment, such
as photodiode array detectors, which will enable large bodies of data to be
rapidly acquired.
The three data sets in Figure 2.3B (the acrylamide quenching of a
mixture of 2-methyl- and 3-methylindole) were actually fitted simultaneously
by Eq. (2.17), using global dynamic and static quenching constants and the
fractional contributions of the components at each emission wavelength. If
only a single data set (single emission wavelength) had been analyzed alone,
the heterogeneity could easily have been missed. This is because the downward
curvature is slight, since the difference between the is not great, and since
static quenching further masks the downward curvature. By linking the three
data sets, the components are easily recovered. We have also applied such
simultaneous analysis of quenching data to a two-Trp-containing protein.(219)
2.6.2. Nonlinear Least-Squares Fits
Practitioners of solute fluorescence quenching are often guilty of the same

lax data analysis practices as are scientists in other areas. Like many other
equations used in biochemistry, the simplest form of the Stern–Volmer equa-
tion is a linear function. Before the advent of computers, scientists favored
linear transformations of equations, and equations like the Stern–Volmer
equation have become traditionally accepted. Actually, a more statistically
sound plot would be of versus [Q]. As shown in Figures 2.1 and 2.26
such a direct plot is nonlinear, but with nonlinear least-squares (NLLSQ)
programs and microcomputers, fits to a direct plot can be obtained very
easily. Acuna et al.(220) and Stryjewski and Wasylewski(221) have described
NLLSQ programs for Stern–Volmer quenching. Both programs will fit
versions of the equation for more than one fluorescent component. In our lab
we have extended the program of Stryjewski and Wasylewski to include
graphics capabilities, record cataloging, and the simultaneous fitting of data at
different wavelengths. Figure 2.26 shows various plots ( versus [Q] and
versus [Q]) of data for the quenching of a protein's fluorescence. Also
shown is a deviation pattern, which demonstrates the goodness of the fit. With
such NLLSQ programs it should no longer be acceptable to use less rigorous
procedures, such as taking the limiting slope of plots of versus 1/[Q],

to analyze quenching data.
2.6.3. Distribution of Lifetimes or Rate Constants
In recent years there has been discussion as to whether the nonexpo-

nential fluorescence decays that are commonly observed for fluorophores in
biochemical assemblies are best described in terms of a small number of
distinct lifetimes or an essentially continuous distribution of lifetimes.(138–141)
We do not wish to fuel this debate here, but we simply raise this issue in order
to discuss the implications in solute quenching studies. If an individual
fluorophore attached to a macromolecule is characterized by two or more
lifetimes (whether these are discrete values or a distribution) and if there is a
single rate constant for the solute quenching of each decay state, then there
will be two or more quenching constants, since Consider, for the
sake of discussion, the pseudo-Lorentzian distribution of lifetimes centered
around 5 ns in Figure 2.27A. A single rate constant (Figure 2.27B) will give a
distribution of values (Figure 2.27C) and this will result in a slightly
downward-curving Stern–Volmer plot (Figure 2.27D). This effect of a lifetime

distribution on Stern–Volmer plots was pointed out by James et al.(139)
Ludescher et al.(66) considered this effect in fitting their data for the iodide
quenching of phospholipase (for which they reported four decay times).
There is, of course, evidence that individual Trp residues in proteins
decay in a nonexponential manner, and the above simulations may be of
general significance. That is, the Stern–Volmer plots for the quenching of
individual Trp residues may not be linear. In actuality, the Stern–Volmer
plots for the quenching of most single-Trp proteins by acrylamide and oxygen
are not downward curving. Such Stern–Volmer plots often curve upward
slightly, due to static quenching or the transient effect discussed in Sections
2.2.3 and 2.3.8. Also, the predicted downward curvature in Figure 2.27D is
barely detectable, even when the width of the distribution of values is very
large.
In Figure 2.29 is shown a Stern–Volmer plot for the acrylamide
quenching of the single Trp in a highly purified sample of cod parvalbumin.
The plot is not perfectly linear. The fluorescence decay of this protein is non-
exponential.(222) A distribution of values was fitted to this data set as
shown in Figure 2.29B. The fit shown is not necessarily better than a fit to two
discrete values, and this model must be given due consideration.
A distribution of values may also result from the existence of a dis-
tribution of quenching rate constants, as shown in Figure 28B and C, even
though the initial fluorescence decay may be a pure exponential. A slightly
downward-curving Stern–Volmer plot is also predicted. A distribution of

values may be due to the existence of different conformations of the protein
(see Section 2.3.1). It is also possible to have a distribution of both lifetimes
and rate constants, which would further broaden the distribution and
produce a greater departure from linearity.
The difference between having a distribution of lifetimes and a distribution
of rate constants is that, in the former case, the solute quenching process will
result in a decrease in the width of the lifetime distribution (Figure 2.27A).
If there is a distribution of values and an initial exponential lifetime,
quenching will result in a distribution of lifetimes (Figure 2.28A).
The above general discussion focuses on the effect of a pseudo-continuous
distribution of lifetimes or values, but also applies when there are two
lifetimes or values for a single fluorophore. These considerations are also
in addition to the transient effects that are discussed in Section 2.3.8.
2.6.4. Experimental Improvements
Many researchers have interfaced fluorometers with personal computers

to acquire steady-state emission data on-line. The use of automated delivery
systems enables quencher- and fluorophore-containing solutions to be mixed
directly in a fluorometric cell. Such systems minimize human handling errors.
Desilets et al.(223) have described a continuous-flow (linear quencher gradient)
system which uses solvent-delivery equipment of the kind commonly used in
liquid chromatography. We are using a computer-controlled syringe-type

dispenser to deliver aliquots of a concentrated quencher solution into a cell
containing the fluorescing solution.
2.7. Phosphorescence Quenching
The triplet state of aromatic chromophores is usually much longer lived

than the lowest singlet state. The phosphorescence lifetime of Trp residues
in proteins, for example, is known to range from to 1 s in the absence
of oxygen at room temperature.(224–226) These long lifetimes have made it
desirable to perform solute quenching studies of phosphorescence. With the

longer lifetimes, it should be possible to measure lower values for triplet
quenching rate constants, (as compared to for the fluorescent state).
For deeply buried chromophores (i.e., Trp residues of alcohol dehydrogenase,
alkaline phosphatase, or apoazurin), it may be possible to better characterize
the kinetic exposure of such groups by their values than by their
values for a particular quencher (since prohibitively high quencher concentra-
tion may be required for fluorescence quenching).
Horse liver alcohol dehydrogenase (LADH) and alkaline phosphatase
(AP) are among the proteins which show long-lived phosphorescence at room
temperature.(227) Their phosphorescence is believed to arise from the deeply
buried Trp residues (Trp-314 and Trp-109, respectively) in these proteins.
Molecular oxygen is the triplet state quencher which has most often been
employed. Of course, the removal of dissolved oxygen is necessary to observe
room temperature phosphorescence of Trp residues in proteins. When oxygen
is added back, a decrease in phosphorescence lifetime results, which enables
to be determined. Because very low oxygen concentrations are needed,
there have apparently been experimental problems regarding the best degas-
sing procedures, means of introducing oxygen, and photodepletion of oxygen
by intense light sources.(228) Consequently, there is some inconsistency in
values for oxygen quenching of Trp residues in proteins. For AP,
Strambini (227) has reported an oxygen as compared
to a value of reported by Calhoun et al.(84) Likewise, there
is over a ten fold difference in the published values of the oxygen for
LADH.(84, 227)
If the low values of found by Strambini are true, then the rate
constants for triplet quenching of these buried Trp residues are very small.
The interest, of course, is to compare oxygen quenching and values
to see if both quenching processes are reported the same degree of exposure
of the Trp residue. LADH and AP have more than one Trp residue, which
makes it difficult to determine the for the most buried residue. Using
double-quenching experiments, the oxygen value for Trp-314 of LADH
(57 )
has been estimated to be For AP, the Stern–Volmer
plot for oxygen quenching of its fluorescence is nonlinear, and it is difficult
to estimate the for Trp-109. Nevertheless, Strambini’s values of to
for the oxygen for these proteins are much smaller than
the estimated for LADH and AP. Strambini discussed
this discrepancy and offered explanations, including the possibility that the
contact distance for fluorescence quenching by oxygen may be larger
than for triplet quenching.
Ghiron et al.(229–231) have monitored room temperature triplet–triplet
absorption of Trp residues in nine single-Trp proteins, following a 265-nm
laser pulse, and have determined values, for oxygen and acrylamide, for
the “quenching” of this triplet absorption. These workers confirmed their

values by making phosphorescence decay measurements for those proteins
with long decay times. While their study did not include LADH and AP, their
oxygen values are more in line with those of Calhoun et al.(84) On
comparing their oxygen values with values, Ghiron et al. found a
(231)
ratio in the range of 0.1 0.6.
With acrylamide, Ghiron et al.(230) also determined values for
several single-Trp proteins. These values were found to range from
to The ratios were found to be as low as 0.001 for
the internal Trp residue of ribonuclease Calhoun et al. had previously
noted that acrylamide (and several other quenchers) has a very low
value for the internal Trp of LADH. These workers
argued that this indicates that such quenchers cannot penetrate into the
matrix of proteins on the nanosecond time scale. However, Ghiron et al. have
pointed out that the acrylamide quenching of indole and N-acetyl-L-tryp-
tophanamide phosphorescence does not appear to be completely efficient (the
efficiency is about 0.3 in water) and that the efficiency of phoshorescence
quenching by acrylamide decreases significantly as the polarity of the solvent
decreases (i.e., the efficiency is 0.002 in acetonitrile). As with the fluorescence
quenching studies with succinimide (see Section 2.3.6), the fact that
acrylamide is not an efficient quencher of Trp phosphorescence in all micro-
environments makes it tenuous to try to interpret the magnitude of values
for buried Trp residues in proteins. Also, Ghiron et al.(230) found the
temperature and viscosity dependence of the acrylamide in proteins to be
very difficult to rationalize. This led Ghiron et al. to cast doubt on the use-
fulness of phosphorescence quenching by acrylamide. Oxygen, on the other
hand, has a quenching efficiency of about for indole phosphorescence,
and this efficiency does not seem to change with solvent.(231,232) These oxygen
values are larger than the predicted spin statistical factor of one-
ninth (231,233,234) ; however, they are consistent with other models for oxygen-
triplet quenching reactions.(235,245) Ghiron et al. also found the oxygen to
not show Stokes–Einstein behavior for the quenching of model lumiphores.(231)
Barboy and Feitelson(236) have substituted a Zn-protoporphyrin into
myoglobin, in place of the nonfluorescent Fe-protoporphyrin. This Zn-
protoporphyrin shows delayed fluorescence, and they have used oxygen and
anthraquinonesulfonate to quench its triplet state. With both quenchers the
quenching rate constant for the protein was found to be about tenfold lower
than that for Zn-protoporphyrin that is free in aqueous solution. Also, the
apparent activation energy for quenching in the protein was found to be
about 6.0 kcal/mol, for both quenchers, as compared to about 3.0 kcal/mol for
the free lumiphore. Thus, the protein matrix shields the protoporphyrin ring,
and Barboy and Feitelson interpreted their results in terms of a “gated”
penetration model.
2.8. Conclusion
Solute fluorescence quenching reactions are a poor man's means to

obtain kinetic information about luminescing systems. The method should
continue to enjoy widespread use in the determination of the accessibility of
fluorophores, and, as discussed in Section 2.5, quenching reactions have
been found to be useful in conjunction with other types of fluorescence
measurements.
The intrinsic luminescence of biological systems is usually complex, and
so quenching data may be complex as well. With nonlinear least-squares
analyses, and especially with simultaneous analyses of linked data sets, it
should be possible to extract meaningful quenching parameters. We look to
improved data analysis procedures to be the most important advance in the
methodology in the upcoming years.
Acknowledgments
I wish to express my appreciation to my mentor, Dr. Camillo A. Ghiron,

for his helpful advice and many stimulating discussions.
Some of the unpublished work presented here was performed under the
support of grant DMB 85-11569 from the National Science Foundation.
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3
Resonance Energy Transfer

Herbert C. Cheung
The mechanism of fluorescence resonance energy transfer (FRET) was first

elucidated by Förster some four decades ago.(1,2) This radiationless transfer
of excitation energy from a donor to an acceptor chromophore is governed
by a long range dipole–dipole interaction, and the Förster formulation
is generally applicable to solution and the aromatic chromophores of bio-
chemical interest. FRET offers an experimental approach to the determination
of molecular distances in the range 10–80 Å through measurements of the
efficiency of energy transfer between a donor and an acceptor located at two
specific sites. Because of the sensitive inverse sixth power dependence of the
transfer efficiency on the donor-acceptor distance, FRET is also a sensitive
technique for detection of global structural alterations.
It has long been recognized that a major uncertainty in the determination
of molecular distances by energy transfer is in the orientation factor for
dipole–dipole coupling. Since this parameter cannot be determined by any
current solution technique, the distances calculated from energy transfer data
usually are not unique except for cases where an appropriate average value of
the orientation factor can be applied. Early energy transfer studies on macro-
molecular systems were largely qualitative in nature, and most of the transfer
data were used to demonstrate structural perturbations. It was not until the
mid-1970s that the potential usefulness of FRET became widely appreciated
and the literature on molecular distances based on energy transfer began to
build up. This transition was due, in large part, to advances in instrumenta-
tion for accurate determination of fluorescence lifetimes and to suggestions
made by several groups to minimize the uncertainty in the orientation factor.
These suggestions included (1) using polarized emission data to define the
mobility of donor and acceptor bound to a macromolecular substrate and to
Herbert C. Cheung • Department of Biochemistry, University of Alabama at Birmingham,

Birmingham, Alabama 35294.
Topics in Fluorescence Speclroscopy, Volume 2: Principles, edited by Joseph R. Lakowicz. Plenum
127
128 Herbert C. Cheung
estimate a range of the orientation factor, (2) choosing donor and acceptor
chromophores that have mixed polarizations and exhibit small limiting
polarization properties, and (3) using statistical interpretations of energy
transfer data to define the limits for the donor–acceptor distance and the most
probable distance.
The Förster formalism of FRET is based on the assumption that the
donor and acceptor chromophores are stationary on a time scale comparable
to the lifetime of the excited state and, as a consequence, the donoracceptor
separation is static and can be described by a single distance. The dynamic
nature of globular proteins and polymers has long been recognized, and the
possible existence of a distribution of the donor–acceptor distances in such
systems was first explored over 15 years ago. Very little progress was made
in the experimental investigation of such distributions for specific donor–
acceptor probes attached to proteins until 1987. The recent progress in this
direction indicates that FRET should be useful in monitoring structural tran-
sitions. Only a few physical methods can yield direct structural information on
macromolecules in terms of molecular distances, and FRET is one of them.
The distribution of energy transfer distances offers a potential experimental
approach to the investigation of the mechanism of protein folding and unfolding
and the intermediates involved, flexibility of nucleic acids, and distribution of
lipids in membrane.
3.1. Long-Range Dipole–Dipole Interaction
Transfer of electronic excitation energy from one atom or molecule to

another may involve different electronic states of both the donor and acceptor
and may be governed by different mechanisms. In all these mechanisms, the
transfer takes place under the condition of conservation of total energy and
thus occurs as a resonance process. The conservation of energy requires that
the energy of the electronic state of the acceptor molecule must be either the
same or less than that of the donor molecule. The excess of electronic energy
after transfer has taken place may be dissipated into vibrational energy.
The transfer process in effect competes with other modes of deexcitation
including direct emission of photons with energy appropriate to the electronic
state of the donor. In this chapter we are primarily concerned with energy
transfer between short-lived singlet electronic states in which (1) the energy of
interaction is classified as very weak, meaning that the rate of transfer is
proportional to the square of the vibronic interaction energy, and (2) the
interaction between donor and acceptor chromophores is purely dipolar so
that overlap between electronic wave functions of the donor and acceptor as
a means of deexcitation is excluded. Under these stringent conditions, the rate
Resonance Energy Transfer 129
of transfer of excitation energy by the weakly coupled resonance mechanism

is given by the Förster formulation (1,2)
where k 2 is the orientation factor for dipole–dipole interaction and is deter-

mined by the angle between the donor and acceptor dipoles, is the
fluorescence quantum yield of the donor in the absence of the acceptor, n is
the refractive index of the medium between the donor and acceptor, N is
Avogadro’s number, is the fluorescence lifetime of the donor in the absence
of the acceptor, R is the distance between the centers of the donor and
acceptor chromophores, and J is the normalized spectral overlap integral,
given by
where is the fluorescence intensity of the donor in the absence of the

acceptor at wavelength and is the molar absorption coefficient of the
acceptor at Equation (3.1) is valid regardless of whether the donor and
acceptor molecules are of the same or different kind and whether or not the
spectra of these molecules have vibrational structure. Equation (3.2) represents
the overlap of the emission spectrum of the donor with the absorption
spectrum of the acceptor, modified by the factor
An alternative expression of the transfer rate is given by
where R0 is the Förster critical distance at which 50 % of the excitation energy

is transferred to the acceptor (50% transfer efficiency). R0 then defines the
spatial relationship of the donor and acceptor chromophores at which the
probability of donor deexcitation by energy transfer equals the probability of
deexcitation by other processes that occur in the absence of the acceptor.
Equations (3.1) and (3.3) can be combined to yield
In Eqs. (3.l)–(3.3) the fluorescence intensity is in arbitrary units, and

the other parameters are expressed in fundamental units: R, R0, and in cm,
and J in cm6/mol. Equation (3.4) can be rewritten as
There are several useful forms of Eq. (3.5):
can be determined from experiments independent of energy transfer. Once

it is determined, the donor-acceptor separation R can be calculated from the
experimental value of transfer efficiency (E) through the expression
Förster’s equation of energy transfer is proportional to and the overlap

integral J and inversely proportional to the sixth power of the donor–acceptor
separation R. By using donor and acceptor chromophores attached to the
ends of fused steroids of a known dimension, Latt et al.(3) showed that the
observed transfer efficiency was in agreement with that predicted by Förster’s
equations. A more quantitative test of the dependence of energy transfer
was provided by Stryer and Haugland, (4) who used a series of oligomers of
poly-L-proline ranging from 12 to 46 and containing a donor and an
acceptor at the ends. They found the observed transfer efficiencies to vary with
the inverse power of R, in excellent agreement with the predicted
value. The dependence was also confirmed by Buecher et al.(5) in
molecular sheet experiments in which the donor and acceptor were separated
by multilayers of fatty acids of known dimensions. Finally, by changing the
magnitude of J over a 40-fold range, Haugland et al.(6) showed that the
transfer rate is in fact proportional to J. These studies have established the
general validity of Förster’s theory, except for the angular dependence given
by
3.2. Determination of Energy Transfer
The transfer efficiency is defined as the ratio of the transfer rate to the
sum of the rates of all deexcitation processes and is given by
where kf is the rate of fluorescence decay, and k' represents the sum of the
rates of all other deexcitation processes. Experimentally, E can be calculated
from either the relative quantum yields (or fluorescence intensities) or
lifetimes of the donor determined in the presence and absence of the acceptor:
where Fd is the donor fluorescence intensity determined at a given wavelength

in the absence of the acceptor, Fda is the corresponding quantity determined
in the presence of the acceptor, and are the donor lifetimes in the
absence and presence of the acceptor, respectively. If the acceptor is also
fluorescent (which is not a requirement for energy transfer to occur), its
emission can also be used to determine E:
where is the absorbance of the acceptor in a sample containing both

acceptor and donor, and is the absorbance of the donor in the same
sample containing both donor and acceptor, both measured at a wavelength
at which the donor absorbs strongly; Fad is the fluorescence intensity of the
acceptor in the sample containing acceptor and donor, and Fa is the acceptor
intensity in the absence of the donor, both excited at the donor wavelength
and measured at an acceptor wavelength This procedure based on enhan-
cement of acceptor fluorescence is considerably more complicated than the
donor intensity method (Eq. 3.13a) because with most donor–acceptor pairs
some direct excitation of the acceptor occurs upon irradiation in the donor
absorption range. It is also possible to determine E from the decay kinetics of
the acceptor.(7) Because of the complexity of the decay pattern, this method
does not appear to have been used with success.
The orientation factor is given by
where is the angle between the emission dipole of the donor and the
absorption dipole of the acceptor, and are the angles between the
vector joining the donor and acceptor and the emission and absorption
dipoles, respectively. k2 can range from 0 to 4.0. The minimum value obtains
when the donor emission and acceptor absorption dipoles are perpendicular
to each other, and the maximum value corresponds to parallel and aligned
dipoles. If both dipoles sample all orientations during the interval of the
excited state (isotropic, dynamic averaging),
3.3. Proximity Mapping of Molecular Assembly
A single distance between two specific sites within a macromolecule

provides very little information on molecular geometry, but several such
distances can be used to define the general shape and dimensions of the
molecule. It is easy to visualize that to specify the relative configuration of
four noncoplanar points requires six distances. This configuration is not
unique because of an unresolvable ambiguity as to whether the fourth point
is above or below the plane defined by the first three points. For each
additional point, four more distances are required to relate it uniquely to
the first four points. Thus, for a total of N points the number of
distances required to determine the relative locations of the specific points by
triangulation is 6 + 4(N – 4) = 4N – 10. This three-dimensional map is
complete except for the handedness alluded to above.
When FRET distances are used to construct a molecular model, several
practical problems must be considered. First, it may not be feasible to
determine all 4N – 10 distances because of labeling difficulties. With protein
assemblies, reconstitution with labeled components may not always be
possible. Second, even if all 4N – 10 distances can be determined, there is
still the uncertainty concerning the orientation factor. Third, the FRET
distance refers to the distance between the centers of two chromophores which
frequently are not intrinsic components of the molecule. If the number of
measured distances is one or two less than 4N – 10, the distances are still
useful and can provide constraints on alternate models. The examples selected
for discussion in this chapter are confined to proteins and protein assemblies.
Individual nucleic acids and membranes will not be covered because of the
lack of space.
3.4. Experimental Strategy
3.4.1. Sample Preparation
An ideal system for FRET measurements is one that contains a single

intrinsic donor chromophore and a single intrinsic acceptor chromophore. An
example would be a peptide containing a single Tyr (donor) and a Trp
(acceptor). Usually, it is necessary to introduce extrinsic fluorophores through
either noncovalent binding or chemical modification of well-defined functional

groups. Chemical modification always poses potential experimental complica-
tions because of (1) possible labeling heterogeneity, (2) possible structural
alteration resulting from the modification per se, and (3) nonstoichiometric
labeling of one or both sites. It is important to minimize these potential
complications by using appropriate control samples. When soichiometric
labeling of both donor and acceptor sites is obtained or when single intrinsic
chromophores are used. Eq. (3.13a) can be used without modification to
determine E by measurements of the steady-state emission intensity of the
donor. If the donor decays monoexponentially, Eq. (3.13b) is also directly
applicable in its simple form.
To use the donor emission properties for FRET measurements, three
separate samples are generally required. A donor-containing or donor-labeled
sample is needed for determinations of donor quantum yield and donor
intensity (F d ) or donor lifetime at a given emission wavelength at which
the acceptor chromophore has negligible or no detectable emission. The
measurement of donor emission properties is then repeated at the same
excitation and emission wavelengths with a second sample which contains
both the donor and acceptor chromophores to obtain the donor intensity
(F da ) or lifetime in the presence of the acceptor. A third sample contain-
ing only the acceptor is required for determination of the absorption spectrum
of the attached acceptor, and for selection of an appropriate emission
wavelength for measurements of donor emission properties. If possible, the
donor-containing sample should also contain a nonabsorbing acceptor
analogue attached to the specific acceptor site. This strategy provides an
assurance that the observed spectral properties of the donor more closely
reflect its properties in the donor-acceptor-labeled sample in the absence of
energy transfer.
3.4.2. Measurement of Transfer Efficiency
An example of a study based on the procedure outlined in Section 3.4.1

is shown in Figure 3.1. We modified the heavy chain of myosin ATPase
subfragment-1 (S-l) at Cys-707 with the donor probe N-iodoacetyl-N´-
(5-sulfo-l-naphthyl)ethylenediamine (1,5-IAEDANS) and at Cys-697 with the
acceptor 5-(iodoacetamido)fluorescein (IAF). Labeling specificity was par-
tially verified by functional tests, digestion of the modified S-l with trypsin
and hydroxylamine followed by gel electrophoresis, and detection of fluorescent
fragments by UV illumination. The separation between the two sulfhydryl
groups (commonly referred to as SH1 and SH 2 ) in the doubly labeled protein
was determined by measurements of the donor fluorescence intensity.(8)
Curve A in Figure 3.1 is the emission spectrum of a sample of S-1 (sample A)
which was modified with the donor at the donor site and labeled at the
acceptor site with the nonabsorbing sulfhydryl reagent N-ethylmaleimide
(NEM). Sample B was an acceptor-labeled protein which was also labeled at
the donor site with NEM. Curve B is the spectrum of this sample. Curve C is
the spectrum of S-1 doubly labeled with the donor and the acceptor. Curve D
is the spectrum of a mixture of samples A and B. This spectrum agrees well
with the sum of curves A and B. The quenching of the donor fluorescence
in the donor–acceptor-labeled protein was accompanied by a concomitant
enhancement of the acceptor fluorescence. These reciprocal spectral changes
are qualitative evidence of resonance energy transfer. Since the labeling of
donor and acceptor was not stoichiometric, Eq. (3.16) was used to determine
the transfer efficiency:
where is the observed degree of labeling of the acceptor site. and

were measured at several emission wavelengths below 475 nm, and the
averages of the intensities from these measurements were used to calculate E.
As a check on the results obtained by the donor intensity method, we
also measured the acceptor enhancement for one series of experiments. The
value of E was found to be 0.64 when determined by the donor quenching
method and 0.60 by the acceptor enhancement method. The close agreement
provided further confidence that FRET was being investigated.
When lifetime is used to determine E, Eq. (3.13b) can be employed if the
donor decays monoexponentially. This method is not affected by non-
stoichiometric labeling. If the degree of acceptor labeling equals or exceeds
that of donor labeling, every donor molecule can be expected to participate in
energy transfer. The lifetime of these donor molecules will be reduced,
but the decay will remain monoexponential. If acceptor labeling is less than
donor labeling, the decay pattern will become biexponential because a
fraction of the donor molecules do not participate in energy transfer. The long
lifetime of the biexponential decay is which is the lifetime of the non-
participating donor molecules, and the short lifetime is The fractional
amplitudes associated with the two lifetimes should approximate the propor-
tions of the two chromophores. Lifetime measurements for energy transfer
studies have the advantage of being independent of concentrations. If lifetimes
can be determined with a high accuracy, E obtained by this method should
have a smaller uncertainty than that obtained by the steady-state intensity
methods. If the donor decay is not single exponential, a weighted average
(e.g., of the lifetimes of the donor is usually taken
as and the corresponding weighted average determined from the sample
containing both donor and acceptor is In this case, stoichiometry labeling
of both donor and acceptor sites will facilitate the analysis.
3.4.3. The Orientation Factor
The orientation factor has been widely discussed in numerous FRET

studies because, except in rare cases, it cannot be uniquely determined in
solution. Early studies on molecular distances were frequently based on the
assumption that both donor and acceptor dipoles randomize rapidly
(dynamic averaging) and sample all orientations (isotropic condition) during
the short interval when energy transfer occurs. Under these conditions,
Frequently, these conditions are not met because the chromophore orienta-
tions are limited by the surrounding macromolecular structure. The resultant
distribution of dipole orientations is likely restricted to a narrow range of
geometries. A variation of from 4 to results in only a 35% error in R,
but a variation from to 0.01 results in a twofold decrease in R. Several
protocols have been proposed to estimate the possible range of or to

experimentally minimize the uncertainty. These will be summarized below
with emphasis on the protocols proposed by Dale and co-workers.(9,10)
3.4.3.1. Estimation of from Depolarization Factors

Polarization spectroscopy provides information on both the orientational
freedom of donor and acceptor chromophores attached to a macromolecule
and on their relative orientations. This information does not normally yield a
unique average value for the orientation factor, but may provide a realistic
estimate of the range of If both donor and acceptor have reorientational
distributions that have at least approximate axial symmetry with respect to a
stationary substrate, average depolarization factors can be derived that are
related to the maximum and minimum values of This relationship is
independent of the particular reorientation distribution functions. In a series
of elegant papers, Dale and co-workers (9,10) have shown that in the presence
of an energy acceptor, the absorption of excitation energy by a donor can be
followed by three depolarizing steps: those due to reorientation of donor and
acceptor and that brought about by energy transfer between them. The
average depolarization factor due to energy transfer is given by the product
of three axial depolarization factors:
where and are the axial depolarization factors for the donor and
acceptor, respectively, and is the axial transfer depolarization factor
associated with the average axial orientations of donor and acceptor. The
axial depolarization factors are related to observable depolarizations by
where is the donor depolarization factor, is the acceptor

depolarization factor, is the experimentally determined limiting anisotropy
of either the attached donor or the attached acceptor, each excited at its
absorption band, and is the observed fundamental anisotropy of either the
free (unbound) donor or free acceptor. The subscripts D and A for the ratio
refer to the donor and acceptor, respectively. The transfer depolariza-
tion factor is determined by
where is the limiting anisotropy of the acceptor determined in the

presence of the donor, and is the fundamental anisotropy of the free
donor, both excited at the same wavelength in the lowest absorption band of
the donor. The axial transfer depolarization factor can be obtained from the
three observable depolarization factors in Eq. (3.17). Dale and co-workers(10)
have published contour plots for obtaining the upper and lower limits of
from observed values of
The limiting values of thus obtained represent the best estimate of the
uncertainty in the relative orientations of donor and acceptor chromophores.
In the absence of additional structural information, the uncertainty cannot be
further reduced.
Frequently, it is not feasible to determine the transfer depolarization
because of extensive overlap between the donor and acceptor emission bands.
When is unknown and if are positive, simple expres-
sions(10) are available for direct calculation of the limits of
If both donor and acceptor do not reorient after excitation, and

are each equal to 1 (limiting anisotropy equals fundamental anisotropy) and
If the donor and acceptor orientations are
completely randomized immediately after excitation (limiting anisotropy = 0),
Thus, for the isotropic dynamic averaging approximation to hold,
the axial depolarization factors must be zero or at least very small (i.e.,
limiting anisotropies very small and approaching zero).
The limiting anisotropy can be determined from the Perrin equation:
where k is Boltzmann's constant, T the absolute temperature, the solvent

viscosity, V the volume of the hydrated macromelecule, and r the observed
anisotropy. The value of r0 is obtained from the intercept in a plot of
versus or merely if the anisotropy measurements are made iso-
thermally. The viscosity is usually varied by addition of sucrose. The lifetime
should be included as a variable because it usually is temperature-dependent
and may also change upon addition of sucrose. The limiting anisotropy is
independent of rotational motion of the macromolecule and reflects orientation
of the attached chromophore immediately following excitation. Alternatively,
this parameter can be obtained in a single experiment from measurement of
time-resolved anisotropy decay, r(t). If the decay is monoexponential, the
anisotropy at zero time, r(0), is the limiting anisotropy. If the decay is

resolved into two components reflecting the rapid local motion (short correla-
tion time) of the attached chromophore and the overall macromolecular
motion (long correlation time), is the amplitude of the anisotropy decay
associated with the long correlation time.
The fundamental anisotropy is an intrinsic property of the
chromophore. It is 0.4 if the absorption and emission dipoles are collinear and
less than 0.4 if the angle between them is greater than 0°:
Since the theory developed by Dale and co-workers assumes single transition
moments, it may be appropriate to use 0.4 for for both donor and acceptor.
However, absorption and emission transition dipoles are in general not
collinear, and the experimentally observed values for a number of
chromophores are indeed considerably less than 0.4. A second origin for
is mixed polarizations; that is, the absorption or emission across a
spectral region is characterized not by a single transition dipole moment,
but by a combination of two or more incoherent dipole moments.(11) For
chromophores with observed it is not entirely clear whether the
observed value or 0.4 is more appropriate for calculation of depolarization
factor. Both values have been used in published studies. (8,12) The use of the
observed value offers a more conservative estimate of because this choice
yields a slightly wider range in than the use of 0.4.
Probes that possess spherical symmetry with triply degenerate transitions
polarized along mutually perpendicular directions approximate isotropic
oscillators. Chromophoric metal ions such as and the lanthanide ions
such as belong to this group. If such a metal ion is
used as either an energy donor or energy acceptor, the uncertainty in is
reduced to 12% or better. If these isotropic probes are used as both the donor
and the acceptor, the uncertainty in is completely removed and
The lanthanides compete for sites in several -binding proteins and
in Ca-ATPase. Their use can yield highly accurate intersite distances for these
systems.
It has been frequently suggested that if both attached donor and attached
acceptor have unconstrained, isotropic, and rapid motions a reversal
of the points of their attachment should have little effect on the isotropic and
rapid motions. To obtain an “experimental” verification of the validity of
the assumption of many investigators in the past performed reverse
labeling with the same donor–acceptor pair in which the donor and acceptor
sites were interchanged. If the interchange did not result in a gross alteration
of the observed value of E, the assumption was then considered valid. This
protocol was used in our previous study (8) of the distance in S-1.
A reversal of the labeling sites produced a very similar transfer efficiency, but
the depolarization factors were large, clearly indicating that the donor and
acceptor were not in randomizing motions. In another study, (12) essentially
identical transfer efficiencies were obtained between two sites in the actin .S-l
complex when the donor and acceptor sites were interchanged. Yet the axial
depolarization factors were above 0.5, leading to a range of 0.155 to 2.999,
again showing that the attached fluorophores were in restricted, not ran-
domizing, motions. For these systems (and possibly some other systems that
have been studied with reverse labeling), the assumption of isotropic and
dynamic averaging was clearly not valid, and it was necessary to describe
the donor–acceptor separation not by a single distance, but by a range of
distances.
3.4.3.2. Statistical Interpretation of

In a treatment that is formally equivalent to that by Dale et al., Haas
et al.(11) have proposed a method to evaluate the range of on the basis of
three-dimensional transition dipole moments. Their procedure also yields
statistical prediction of the most probable distance. They also showed that
chromophores that have mixed polarizations and are characterized by multiple
dipole transitions can have a narrow range. For such chromophores the
assumption of may be more acceptable than for those with a single
transition dipole. Chromophores with the dansyl moiety (e.g., IAEDANS)
exhibit mixed polarizations across the absorption band, and in some FRET
studies involving such chromophores it has been assumed that the range is
likely narrow and Such an argument has been used to justify, at least
in part, the use of with these probes. It was recently pointed out (14) that
the reduction of the range by this mechanism would be possible only if the
acceptor chromophore has multiplicity in its absorption transition dipole.
Multiple transition dipoles in the donor are not likely to affect the range
mainly because directly excited vibronic states are rapidly relaxed to the same
lowest energy level of the excited singlet state before emission occurs. It would
seem that such a donor always emits from a single emission dipole. Further
work will be required to address this problem.
Statistical treatments were developed for frozen but randomly oriented
donors and acceptors, each characterized by a single transition moment.
These methods (15,16) enable calculations of the most probable donor-acceptor
distance from an observed transfer efficiency. The validity of these statistical
approaches has been questioned(10) on the grounds that the orientations of
attached chromophores are generally constrained by the surrounding environ-
ment. It remains an open question as to whether such statistical methods can
yield physically meaningful distance parameters.
3.5. Selected Applications
3.5.1. Myosin and Actomyosin
The first contractile protein studied by energy transfer was myosin

ATPase.(17) We determined the transfer efficiency from the tryptophanyl
residues to an extrinsic probe (8-anilino-l-naphthalenesulfonate) bound to a
single hydrophobic site by measurement of the quenching of the donor emission.
No transfer distance was calculated because of the multiple donors and
because of the uncertainty at that time of the number of tryptophanyl residues
in the enzyme. A small time-dependent change in the transfer efficiency was
detected upon addition of ATP, and this change was correlated with ATP
hydrolysis. These early results suggested that the conformation of myosin
during the steady-state ATP hydrolysis was different from that generated
by mixing myosin with ADP.(18) The existence of more than one myosin
conformation in the myosin ATPase pathway was subsequently elucidated by
other spectroscopic and rapid kinetic methods.
Several lines of physical evidence suggested that there was no significant
change in the gross shape of myosin ATPase subfragment-1 (S-1) during
interaction with nucleotides or actin. This finding and other considerations
have led to the notion that an important aspect of the energy transduction
mechanism in muscle involves communication between functionally important
sites located in S-l, namely, the ATPase and actin sites. This interactive
binding of substrate and actin can lead to localized changes in the S-l
structure. A detailed investigation of such a model will require full knowledge
of the three-dimensional structure of S-l. While crystallography will ultimately
yield the structural information, X-ray data from single crystals will not be
available for some time. FRET studies can provide preliminary information
on the approximate arrangement of selected points.
The heavy chain of myosin S-l contains two reactive thiols, which are
commonly referred to as (Cys-707) and (Cys-697). Chemical
modification of results in a three- to fourfold activation of the
Ca-ATPase activity with a concomitant loss of the EDTA-ATPase and actin-
activated ATPase activities. The additional modification of abolishes all
enzymatic activities. We(8) used the donor intensity method to determine the
energy transfer distance between the two thiols with 1,5-IAEDANS attached
to as the energy donor and IAF linked to as energy acceptor
(Figure 3.1). Very similar results were obtained when was labeled with
the acceptor and with the donor. The following distance parameters
were obtained: is the
distance based on and R(max) and R(min) are the maximum and
minimum distances, respectively, based on calculated
from Eqs. (3.21) and (3.22). The presence of actin shortened the distance by
whereas MgADP had no effect on the distance. A different acceptor,
N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM), was also used to
modify The distance from 1,5-IAEDANS at to DDPM at
was determined by the lifetime technique using phase fluorometry (19) and
pulse fluorometry.(20) This second donor–acceptor pair yielded a value of
27–28 for shorter than from the 1,5-IAEDANS–IAF pair. The
1,5-IAEDANS–DDPM distance in S-l decreased by 7-8 upon addition of
MgADP, demonstrating a perturbation of the localized structure by
nucleotide binding. The difference in structure between the two acceptors may
be responsible for the different results.
Of the five cysteines in actin, the penultimate residue (Cys-374) is
accessible for chemical modification under mild conditions. In the monomeric
form (G-actin) the protein contains a single bound ATP. Upon polymeriza-
tion to F-actin, the bound ATP is split, converting the bound nucleotide to
ADP. We previously reported that replacement of bound ATP in G-actin by
1, -ethenoadenosine triphosphate did not impair the ability of actin
to polymerize (21) and determined the distance from bound (donor) to
Cys-374 modified with 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitro-
benz-2-oxa-l,3-diazole (IANBD) as the acceptor to be has
also been used by other investigators to determine the separation between
the nucleotide site and Cys-10 and between the nucleotide site and two
specific points on the myosin S-l heavy chain in the acto · S-1 complex.
Figure 3.2 is a space lattice for S-l and acto · S-1 constructed from FRET
distances.(23) The seven specific points in S-l include three residues on the
heavy chain, one residue on each of the two light chains, and two points
within the nucleotide binding site that is involved in ATP hydrolysis. Ten
distances have been reported between these seven points. Two distances in
actin and eight distances across actin and S-l in acto · S-1 are also included.
The distances shown in Figure 3.2 are far short of the 4N – 10 requirement for
complete spatial specification of acto · S-l or the individual proteins, but have
already provided some important insight into the transduction mechanism.
Filamentous actin (F-actin) is comprised of two strands of actin polymer
that are assembled into a suprahelical structure. Individual actin monomer
(G-actin) is a single-polypeptide protein which is bilobar. The orientation of the
monomer in the filamentous structure based on FRET was first investigated
by Taylor et al.,(38) who measured the transfer between chemically equivalent
sites located on different monomeric units within the suprahelical structure.
In this study two preparations of labeled G-actin were used: one preparation
in which Cys-374 was labeled with IAF serving as the energy donor, and the
other in which the same residue was labeled with iodoacetamidoeosin or
tetramethylrhodamine serving as the energy acceptor. Copolymers of pure
actin filaments were obtained by using various molar ratios of the donor-
labeled and acceptor-labeled G-actins. From the known geometry of the

F-actin filament, which was approximated as a 13/6 helix, and the FRET data
obtained from lifetime measurements, the radial coordinate of the modified
residue was calculated to be about Since this coordinate is the per-
pendicular distance between the residue and the helix axis, the result indicates
that Cys-374 is located near the outer surface of the filament. Three additional
radial coordinates have since been reported: Cys-10(39) and the nucleotide-
binding site,(40) both obtained from donor steady-state intensity, and Gln-41,
determined from time-domain lifetime data.(41) The latter study also showed
from a theoretical analysis that, within certain symmetry limits, four radial
coordinates (N = 4) and six intramolecular distances (4N – 10 = 6) are
required to completely define the orientation of the actin monomer in the
filament. In the same study Kasprakz et al.(41) also found that myosin S-l
binding to F-actin increased the radial distance of Gln-41 from
but had negligible effect on the radial distances of Cys-374 and the nucleotide-
binding site. These results do not rule out conformational changes which may
be induced on actin monomer in the region of the nucleotide site or Cys-374
by interaction with S-l, but probably can eliminate certain models of inter-
action which would require global rotation of actin monomer with large
amplitudes. Of special interest is the effect of binding of activator calcium to
regulated actin filament (actin filament decorated with a full complement of
the regulatory proteins troponin and tropomyosin) on the radial coordinates.
We (Censullo and Cheung, unpublished results) have recently shown that
the radial coordinate of Cys-374 decreases by 9% when the
regulatory proteins are incorporated into the actin filament. In response to
calcium binding the coordinate decreases further to suggesting a
compression of the actin filament. While four radial coordinates have been
reported, complete characterization of the orientation of actin monomer
in the filament must await additional intramolecular distances because
4N – 10 = 6 for this system and only two such distances are at hand (see
Figure 3.2).
The method developed by Taylor et al.(38) was intended for the use of
FRET to investigate actin assembly and disassembly during motile events in
nonmuscle cells. The authors also showed that the emission decay of the
labeled actin microinjected into living Chaos carolinensis was essentially
unaltered and unquenched by the cytoplasm. This observation suggested the
possibility of quantitative measurements of energy transfer in living cells.
FRET was also used recently to follow assembly of synthetic myosin thick
filaments and demonstrate exchange of myosin molecules between the
filaments.(42) Donor (IAEDANS)-labeled and acceptor (IAF)-labeled myosins
were preincubated in 0.5 M KCI. The assembly into filaments was initiated by
reduction of ionic strength by dilution and monitored by following the
decrease of donor intensity. From these data the concentration of myosin that
remained unassembled (critical concentration) was calculated and found to be
40 nM in the range of myosin concentration This determination
was possible because of the high sensitivity of the FRET assay and the
measurement was considerably easier to carry out than that based on analyti-
cal ultracentrifugation. Dynamic exchange of myosin molecules between thick
filaments was demonstrated by following the quenching of donor intensity
resulting from donor-labeled thick filaments and acceptor-labeled thick
filaments. The extent of exchange was found to be 75% after 180 min and was
independently confirmed by using 125I-labeled myosin and ultracentrifugation.
These approaches are applicable to investigations of macromolecular

assembly and disassembly in other systems.
The hydrolysis of ATP by actomyosin ATPase can occur via both the
dissociating and nondissociating pathways, in which the hydrolytic step
occurs on myosin subfragment-1 (S-l) that is dissociated from actin or
attached to actin, respectively. The dissociating pathway can be represented
by
where A is actin and A · S-1 (acto · S-1) is the complex formed between actin
and the myosin heads (S-l). The question of which chemical step is coupled
to force generation during muscular contraction has been extensively
investigated. It is believed that S-l attaches to actin in two states. Weakly
bound states (e.g., A · S-l · ATP, A · S-1 · ADP · Pi ) are associated with the
beginning of the power stroke, and the strongly bound states (A · S-1,
A · S-1 · ADP ) are associated with the end of the power stroke. Transition
from the weakly bound states to the strongly bound states may be accom-
panied by large-scale structural changes within the acto · S-1 complex. These
changes are particularly relevant in deciding whether the myosin head or a
portion of the myosin rod is the energy-transducing element that is involved
in converting chemical free energy to mechanical work. The structures of the
strongly bound states have been extensively studied by various techniques,
but not much is known about the weakly bound states. To investigate this
second attached state requires a method to arrest the contractile process at
the pre-power stroke, where S-l is weakly attached to actin. One way to
obtain stable analogous of the weakly bound state (A · S-1 · ATP) is through
trapping of ATP at the active site by cross-linking the thiols
When distance 14 between actin Cys-374 and S-1 light-chain Cys-177
(Figure 3.2) was measured with non-cross-linked S-l (A · S-1, A · S-1 · ADP),
was found to be in the range When cross-linked S-l was used
to mimic the weakly bound species (A · S-1 · ATP), This
large decrease has been taken as direct evidence for a change in the structure
of myosin heads that could account for tension generation. Qualitatively
similar, but less pronounced, results have also been reported recently on
distance 10 between actin Cys-374 and S-1 Cys-707 by using acto · S-1
that was cross-linked by a carbodiimide in the presence of ATP.(31) was
for non-cross-linked acto S-1 (rigor) and increased to 54 Å for cross-
linked acto · S-l. Not resolved in these studies, however, is to what extent the
observed distance change occurs within S-l as opposed to being due to
reorientation of S-l relative to the actin filament. There is no information on
the kinetics of the distance change that occurs during the transition from
species associated with the pre-power stroke to those associated with the end
of the stroke.
3.5.2. Troponin Subunits
Troponin consists of three nonidentical subunits: troponin C (TnC),

troponin I (TnI), and troponin T (TnT). In vertebrate skeletal and cardiac
muscles these proteins and tropomyosin play a key role in the regulation by
of actomyosin ATPase and the contractile cycle. The initial molecular
event in the regulation is binding of to TnC. The signal is then
transmitted to specific sites on actin via the three other regulatory proteins.
FRET has proved a useful tool for understanding the mechanism of this signal
transmission on the basis of global structural perturbations induced in the
proteins by binding. Our strategy was to measure the separations
between specific sites in each of the troponin subunits and across the subunits
in reconstituted troponin. In one study (43) four specific residues were selected
for the complex TnI · TnC: two (Cys-133 and Trp-158) in TnI and two
(Met-25 and Cys-98) in TnC. These four points yielded six distances. The
Trp in TnI served as an intrinsic donor and the other three residues were
modified by three extrinsic probes: dansylaziridine (DNZ) for Met-25 and

1,5-IAEDANS and IAE for Cys-98 and Cys-133. The donor intensity method
was used to determine the transfer efficiencies for all six distances, and the
lifetime method was used to confirm the results obtained from donor intensity
for one of the distances. A tentative proximity map was constructed from the
six measured distances for TnI · TnC (Figure 3.3). In Table 3.1 are listed the
values for the distances that were determined in the presence of EGTA,
Skeletal TnC has four sites. Two sites bind with a high
affinity also binds competitively at these sites.
The other two sites bind specifically with a low affinity
Since the intracellular concentration is in the millimolar
range, the two high-affinity sites are likely occupied by in relaxed
muscle, where the cytosolic level is in the submicromolar range.
activation involves binding of to the two low-affinity sites. The FRET
distances determined in the presence of and provide a basis for
understanding structural changes that are induced when muscle is activated
by With the exception of the intersubunit distance C(25)-I(158), the
other five distances were significantly perturbed when the ionic medium was
changed from to The changes were in both directions in the
range of 5–10Å . A decrease of 5 Å in C(98)–I(133) in fully reconstituted
troponin was also observed(44) when the medium was changed from to
Although there are inherent uncertainties in the individual measured
distances, large changes in the distances must reflect substantial structural
perturbations. These results suggest large-scale movements of two regions in
each protein toward or away from each other when muscle is switched on.
These movements must play a role in the transmission of the signal.
What is not resolved at this time is whether these changes occur with time
constants that are compatible with the known kinetics of force generation.
The crystal structure of TnC is of considerable interest. It shows the
protein to be dumbbell-shaped,(45) with the N- and C-terminal regions folded
into two globular domains. The two domains are connected by a nine-turn
(32 residues). The middle third of this long helix is not in contact with
other regions of the molecule and is exposed to solvent. Since the protein
crystals were obtained at the question arises as to whether the protein
is also dumbbell-shaped at neutral Our previous study(46) showed that
for the distance between Met-25 (labeled with DNZ) and Cys-98 (labeled
with IAE) was 39 A The actual separation between the points of
attachment is likely smaller because of the size of the probes. When the
was reduced from 7.5 to 5.0, the transfer efficiency was reduced 13-fold,
corresponding to an increase of the donor–acceptor separation by a factor of
2.(47) These results are interpreted in terms of an acid-induced dimerization of
the protein,(48) and suggest that the conformation of TnC in neutral solution
may be different from that predicted by crystallography.
3.5.3. Ribosomal Proteins
In early studies(49) the proximity of the component proteins of the 30S

Escherichia coli ribosomal particle was estimated by using 1,5-IAEDANS as
the energy donor and fluorescein isothiocyanate (FITC) as energy acceptor.
Individual proteins were labeled either at a specific site or randomly on
the protein surface. Each donor-labeled protein was reassembled in parallel
experiments into two 30S samples, one containing no other labeled protein
and the other containing a single acceptor-labeled protein. Transfer efficien-
cies between 20 pairs of proteins were determined by measurement of donor
intensity. Sufficient transfer data were collected to yield 12 distances (15–78 Å)
on a subset of six of the 30S proteins (S4, S13, S15, S16/17, S19, S20). Since
4N – 10 = 14 for this case, two additional distances would be required to
specify the geometric arrangements of the six proteins within the 30S subunit.
By assuming the proteins to be reasonably spherical and the probe dipoles to
be isotropically and randomly oriented, the investigators limited the arrange-
ment of the proteins to four possible models and concluded that the six
proteins were not coplanar. The center-to-center distance between S7 and S9
was 31 Å, in agreement with the value of deduced from neutron
scattering. More recent FRET studies involved labeling specific sites such as
sulfhydryl groups and the 3´ end of RNAs. These studies included (1) the
distances between a thiol group of the elongation factor EF-Tu from Thermus
thermophilus bound to reconstituted Escherichia coli ribosome and the 3´ ends

of I6S RNA, 5S RNA, and one of the two cysteines of Sl, (51) and (2) the
distances from the 3' end of 16S RNA to S1 and S21.(52) In the latter work,
three probes were used: 3-(4-maleimidylphenyl)-4-methyl-7-(diethylamino)-
coumarin (CPM) as energy donor, and fluorescein-5-rnaleimide (FM) and
fluorescein thiosemicarbazide (FTS) as acceptors. The distance between
CPM-S21 (the single cysteine at position 22) and FTS-16S RNA at the 3´ end
was 51 Å, the distance from FTS-16S RNA to CPM-S1 (at one of the two
cysteines) was 68 Å, and that between CPM-S21 and FM-S1 labeled at both
Cys-292 and Cys-349 was 68 Å. Since there were two acceptor sites in S1, the
actual distance from the donor in S21 to either of the two acceptors in S1
would be longer than 68 Å, dependent upon their geometric relationship with
respect to the donor. Binding of poly(uridylic acid) to the 30S subunit
increased the S1–16S RNA distance to at least 80 Å and the S21–16S RNA
distance to 56 Å, but had no effect on the S21–S1 distance. The effect of the
50S subunit on the first distance was negligible, but an increase of 8–10 Å was
found for the other two distances. The combined effect of poly(U) and 50S
was an increase of all three distances in the range of 10–12 Å. In spite of the
fact that poly(U) is known to interact with S1 in 30S subunits, the interaction
leaves the S21–S1 distance unperturbed. The other results suggest that 50S
subunits affect the conformation of S21 in 30S subunits.
Among the proteins of the large SOS subunits, FRET has been used to
determine the distance from L11 to L6 (46 Å) and from L11 to L10 (56 Å).
These results(53) suggested that the L6/L10/L11 domain is tightened during
activation. Energy transfer data(54) were recently used to locate the position of
the single –SH group (Cys-38) of L11 in the E. coli ribosome. The distance
from the –SH group labeled with CPM to the 3´ end of 5S RNA labeled with
FTS was 76 Å and that to the 3´ end of 23S RNA was 69 Å. From these and
other distances within the 30S subunits and between the 30S and 50S sub-
units, it was concluded(54) that L11 is located in the 50S subunit below the
lateral protuberance characterized by L7/L12.
3.6. Comparison of FRET Results with Results from Other Techniques
3.6.1. Comparison with Crystallographic Data
3.6.1.1. Use of Lanthanide Ions

Because of the uncertainty in and because of the finite size of donor
and acceptor chromophores, comparison of FRET distances with those
derived from other methods is generally hazardous. However, the uncertainty
in can be eliminated if both donor and acceptor chromophores are

approximate isotropic oscillators. A number of reports have appeared in
which lanthanide ions were used as replacements in the determination
of distances between metal-binding sites in several -binding proteins.
Such results for four -binding proteins given in Table 3.2 can be
compared with the corresponding distances obtained from crystallography.
The agreement is excellent in two cases, but only reasonable in others. In the
first studies of this kind, (55,56) the FRET distances determined with as
the donor were as much as 3 Å shorter than those determined with as
the donor for the same sites. The possibility was raised by the investigators (55)
that as a donor would in general sense a higher transfer efficiency than
150 Herbert C. Cheng
This effect was particularly pronounced with the pair.

The more recent studies of troponin and calmodulin(59) did not show
a systematic trend for anomalous transfer efficiency that was sensed by
, although the FRET distances were smaller than
It should be noted that in the crystals, lanthanide ions are not always
located at positions identical to those for bound . For TnC, and
located at site III were within 1 Å of the position, but the same
ions bound to site IV were 5.5 Å away from the positions.(45) In a more
(63)
recent study based on a higher resolution (2.2 Å), the distances between
and lanthanide ions in sites III and IV were
refined to 0.3 and 0.8 Å, respectively. These results could very well account for
the 2-Å difference between the interlanthanide and intercalcium distances even
if fluctuations in intersite distances arising from protein dynamics is assumed
to be negligible. The difference between and the shorter observed
for parvalbumin and thermolysin was interpreted (64) as indicating that
exchange interactions involving electron overlap (which leads to a higher
transfer efficiency than dipole–dipole interaction) became important for
transfer between lanthanide ions approximately 11.8 Å apart. This interpreta-
tion was based on a general consideration(64) for the relative contributions of
the two types of transfer mechanism involving chelated and
However, the results shown in Table 3.2 are insufficient for such a generaliza-
tion at this time.
3.6.1.2. Energy Transfer from Tyrosyl to Tryptophanyl Residues
Comparison of FRET distances with crystallographic or other data is

more difficult with aromatic chromophores (either intrinsic or extrinsic) than
with isotropic oscillators. The distance between the two tyrosyl residues
(residues 99 and 138) of calmodulin was reported(65) by measuring the
transfer efficiency from one Tyr to the other, which was nitrated with
tetranitromethane. The best estimate of this separation (from Tyr-99 to the
nitro moiety of nitrotyrosine-138) was for -saturated
calmodulin. The center-to-center distance between the two phenol rings is
11.27 Å.(62) The presence of a nitro group attached to the phenol ring as the
acceptor may, in part, account for the longer FRET distance, but the limited
mobility of one or both phenol rings also may contribute to the observed
transfer distance.
The lens protein calf gamma-II crystallin offers an interesting example. It
contains 15 Tyr and 4 Trp residues. The transfer efficiency from the tyrosyl
residues to the tryptophanyl residues was found to be at
On the basis of the crystal structure, all Tyr–Trp distances were calculated
between the centers of the two aromatic rings; they ranged from 5 to 30 Å.
From these distances and the assumption of the transfer efficiency was
predicted to be 83%, (66) in reasonable agreement with the value observed in
solution. The difference almost certainly arises from the possibility that some,
if not many, of the donor and acceptor chromophores have limited motional
freedom. Since only the short distances contribute significantly to E because
of the dependence, the question is whether the side chains of the residues
that are separated by short distances are highly polarized or depolarized. This
question cannot be answered without other types of information.
A final example is the C-terminal octapeptide of cholecystokinin,
(Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe- ). NMR studies(67) showed that
exists preferentially in a folded conformation with and turns
around the sequence Gly-Trp-Met-Asp and Met-Asp..Phe- . This folded
conformation results in a structure in which the side chains of Tyr and Trp
must be pushed away from each other, resulting in a relatively large separa-
tion between them. This structural feature was confirmed by energy transfer
measurements,(67) which yielded The choice of for this
case is reasonable because indole (acceptor) is characterized by two linear
transition moments and the Trp in the peptide is relatively free to rotate, as
demonstrated by NMR results.
3.6.1.3. Transfer between Aromatic Probes

The unfolding in the N-terminal region of RNase A was recently studied
by FRET(68) from a donor (ethylenediamine monoamide of 2-naphthoxyacetic
acid) attached to carboxyl groups of Glu-49 and Asp-53 to a nonfluorescent
acceptor (2,4-dinitrophenyl) on the -amino group. Since Glu-49 and Asp-53
are close in both the sequence and the X-ray structure, heterogeneity in donor
labeling was unimportant. The interprobe distance was found to be
A under folding conditions. The contributions of the finite size of the
donor and acceptor moieties to the transfer distance were estimated by
combining the conformational statistics of the donor linkage with geometrical
considerations. These contributions were then combined with structural
information from unmodified RNase to give an estimated average interprobe
distance of 36 Å. This approach provides an expedient comparison of FRET
results with X-ray structural information. The excellent agreement in this case
justified the use of
3.6.2. Comparison with Cross-Linking Data
Chemical cross-linking of specific groups by bifunctional reagents is

generally taken as evidence for close proximity between the two groups. Their
average separation, however, may or may not correspond to the span of the
bifunctional reagents. The FRET distance between the two thiols and
of myosin subfragment-1 (distance 6, Figure 3.2) is in the range of
30–45 Å, dependent upon which acceptor is used. It has been known that the
same two thiols can be cross-linked by a variety of bifunctional reagents with
widely different spanning lengths (2–14 Å). The segment of the polypeptide
chain between the two thiol groups is thought to be flexible. There must exist
a wide distribution in the separations between the two –SH groups. FRET
measurements yield an average of these distances. The average contains not
only a population weighting, but also weighting by the inverse sixth power of
the distances, which favors shorter distances. The cross-linking experiments
investigate instantaneous separations that fall into a certain narrow range,
and the method has a very narrow distance window, whereas FRET results
reflect the entire distance range.
Another example is the TnI • TnC complex. The transfer distance in the
presence of between Cys-133 of TnI and Cys-98 of TnC is in the range
of 41–74 Å.(43) These two groups have been cross-linked by l,3-difluoro-4,6-
dinitrobenzene in the presence of , (69) providing evidence for their very
close proximity. Even allowing for the finite size of the donor and acceptor
probes, it is unlikely that the average transfer distance could be as small as
10 Å. The binding constant(70) of the labeled TnC for unlabeled TnI or of the
labeled TnI for unlabeled TnC is in the presence of
The proteins are not very tightly held together. Some conformational fluctua-
tions within each protein and in the complex as a whole can be expected.
In consequence, the FRET distance is an average value. These examples
illustrate the inadequacy of using a single average distance to describe a
proximity relationship. Section 3.9 will address the experimental determina-
tion of distribution of FRET distances from both lifetime and steady-state
data.
3.7. Application of FRET to Enzyme Kinetics
Many enzymes contain tryptophanyl residues. If their substrates carry

chromophores that can accept excitation energy from Trp, energy transfer can
be expected between enzyme and substrate as the enzyme-substrate (ES)
complex is formed. Formation and breakdown of the ES complex can be
directly monitored in stopped-flow experiments at submicromolar enzyme
concentration. First proposed in the early 1970s, this kinetic approach to
resolve mechanistic details of catalysis has been developed mainly by Auld
and co-workers (71,72) during the past decade and applied to a number of
proteases with dansylated substrates. Both pre-steady-state and steady-state
kinetics can be observed in one experiment. Analysis of the pre-steady-state
kinetics enables determination of the number of intermediates and individual
rate and binding constants.(73) Direct observation of the ES complex by

energy transfer under steady-state conditions simplifies and supplements
conventional initial-rate kinetic studies. The method is sufficiently sensitive
to allow measurements to be carried out under the kinetic conditions
and A steady state is thus achieved rapidly under
stopped-flow conditions and maintained until the reaction is complete.
The time-dependent fluorescence changes for the binding and hydrolysis
of by chymotrypsin are shown in Figure 3.4. Curve A
shows the change of acceptor (dansyl) fluorescence intensity, which rapidly
reached a maximum, indicating attainment of the steady state. This rapid,
pre-steady state was followed by a slower decay of the fluorescence as the
substrate was hydrolyzed. Reciprocal changes were observed (curve B) when
the tryptophan (donor) emission of the enzyme was monitored. Also shown
in Figure 3.4 are schematic illustrations of the steady-state portion of the
kinetic tracing. The acceptor emission at time zero when the steady state is
achieved is and at any time t thereafter is The area described by the
entire stopped-flow trace is
If the fluorescence properties of the acceptor are unchanged in substrate and

product, is proportional to the steady-state concentration of ES and the
area A under the curve is inversely proportional to the maximum catalytic
rate (turnover number), The reaction velocity during the steady state is
related to and by where is the initial substrate
concentration. The Michaelis–Menten equation can be expressed in terms of
the two stopped–flow parameters:
where is the Michaelis–Menten constant. The steady-state kinetic

parameters can be determined from stopped-flow traces such as those shown
in Figure 3.4 in several ways: (a) from measured values of and
(b) from the dependence of on substrate concentration, and (c) from the
dependence of on substrate concentration at time and at
any time t can be determined directly from kinetic traces similar to that
shown in Figure 3.4, and can be readily determined from the area under
the kinetic trace when the initial concentrations of enzyme and substrate are
known. Thus, a double-reciprocal plot of versus can be con-
structed from the kinetic trace obtained from a single substrate concentration.
The values of and obtained by this method for the hydrolysis of
by chymotrypsin are in excellent agreement with
those obtained by the usual initial–rate method.(72) The action of an inhibitor
can be readily visualized. A competitive inhibitor reduces and does not
affect the area under the kinetic trace. In contrast, a noncompetitive inhibitor
is expected to increase the area A but has no effect on
The rapid fluorescence change during the pre-steady-state time interval
displayed in Figure 3.4 is shown in Figure 3.5 on a faster time scale. During
mixing and within the first 3 ms an increase in acceptor fluorescence
occurred. The subsequent exponential increase to the final level,
was characterized by the rate constant This two-step change reflects
the presence of two intermediates, and these results are consistent with the
accepted acyl intermediate mechanism for ester hydrolysis by chymotrypsin as

represented by the usual scheme
The initial increase in fluorescence to signals the formation of ES, and the
exponential increase to reflects conversion of ES to the acyl intermediate
EA. This example illustrates how both pre-steady-state and steady-state
kinetic parameters can be extracted from one experiment on the basis of
energy transfer.
Several anions including chloride, sulfide, and carboxylate have been
known for over 30 years to inhibit carboxypeptidase A (CPA). The nature of
anion inhibition has been difficult to investigate partly because of the limited
solubility of CPA in the absence of salt and hence the necessity to work with
fairly high concentrations of anions. This difficulty has recently been over-
come(74) by using dansylated di- and tripeptides. Chloride was found to
be a competitive inhibitor as qualitatively indicated by the changes of the
shape of the stopped-flow kinetic traces and by detailed analysis of the traces.
Since for chloride inhibition was strong (40–120 mM) and not strongly
pH-dependent, the investigators concluded that the site of anion interaction is
unlikely to be the metal atom.
3.8. Time-Resolved Energy Transfer Measurements
A major structural information that can be obtained from energy transfer

studies is global perturbation resulting from ligand interactions. Such infor-
mation provides a static picture of what has happened after the interaction.
An important question is whether the perturbation is kinetically competent to
be a component of an overall biological mechanism. Up to this time, very
little attention has been paid to the kinetics of energy transfer changes.
As we have discussed, the distance between the two thiols, and
in myosin subfragment-1 is sensitive to the presence of MgADP. Th transfer
efficiency between the donor (IAEDANS) attached to and the acceptor
(DDPM) linked to was increased upon addition of nucleotide, resulting
in a decrease of the donor–acceptor distance by 7–8 Å. We(75) have
investigated the kinetics of this energy transfer change in stopped-flow
experiments by rapidly mixing the doubly labeled S-l with
MgADP at 20°C. Since the acceptor was not fluorescent, the reaction was
monitored by following the decrease in donor fluorescence. Steady-state
measurements had shown this decrease to be about 15%. Kinetic traces could
not be fitted with a single exponential, but were well fitted with a two-
exponential model. The two apparent first-order rate constants increased

fairly smoothly with increasing ADP concentration and appeared to level off
beyond 1 mM ADP. At the highest ADP level (0.94 mM) studied, was
and was When extrapolated to zero ADP concentration,
was ca. and was The initial slope of the versus [ADP]
plot was and that of the versus [ADP] plot
The anisotropy of the attached donor was also monitored in stopped-

flow experiments over the same time interval used in the intensity exeriments.
In all cases the donor anisotropy remained essentially unaltered during the
change in donor intensity, with an average value that was in agreement with
that determined with the steady-state fluorometer. These results suggest that
there is no change in probe mobility during the course of reaction and
indicate that the observed increase in energy transfer is due entirely to a
biphasic decrease in the donor–acceptor distance from about 29 to 22 Å.
The amplitude of the fast phase, at high nucleotide concentration, accounts
for approximately 70% of the total change of intensity. If the Förster critical
distance remains constant during the reaction, the fast phase reflects a
decrease in donor–acceptor distance of 4–5 Å and the slow phase an additional
decrease of about 2 Å. The use of FRET in conjunction with anisotropy
signals in stopped-flow studies to investigate the kinetics of structural pertur-
bations has advantages over the use of signals from single chromophores. The
amplitude changes associated with the signal from a single chromophore
provide little useful information, but the amplitudes of the change in the
FRET signal can in principle be related to physical changes. Because the
transfer efficiency depends on the inverse sixth power of the donor–acceptor
separation and on the relative orientation of the donor and acceptor, small
structural perturbations can be more readily detected by this method than
with single chromophores. In the present case the physical changes induced
by nucleotide binding, naemely, the movement of different domains of S-l
toward each other, have been time-resolved. In a previous study Garland and
Cheung (76) showed that, on the basis of the emission of single fluorophores,
the kinetics of the interaction of myosin S-l with nucleotides is best described
by a sequential three-step mechanism in which formation of the protein–
nucleotide complex is followed by two first-order steps. The physical origin of
these steps was undefined. The recent kinetic results based on time-resolved
FRET measurements of the same reaction are kinetically compatible with the
three-step mechanism and may provide a physical basis for the previously
proposed reaction scheme.
While the work described above was in progress, the first full report (77)
on time-resolved stopped-flow measurements of energy transfer changes
appeared. The three tRNA binding sites (P, A, E) on E. coli ribosomes have
been investigated by various methods. The separation between the tRNA
anticodon loops in the P (peptidyl) and A (aminoacyl) sites was estimated by

using wybutine as the donor and proflavin as the acceptor, both located to
the anticodon of This distance was 23 Å. (78) A similar method was
used to determine the separation of tRNA bound to the E site from that
bound to the P site (34 A) and the A site (42 Å). These results describe the
tRNA topography in the pre-translocative and post-translocative complexes.
A more interesting problem is determination of the topography during trans-
location. This was accomplished in stopped-flow experiments. Pre-translocative
complexes were prepared by binding and at
the P and A sites, respectively, of poly(U)-programmed ribosomes. These
complexes were then rapidly mixed with elongation factor G (EF-G) and
GTP. Since the tRNA–tRNA distance in post-translocative complexes was
longer than the distance in pre-translocative complexes, a time-dependent
decrease in energy transfer between wybutine and proflavin in the two tRNA
molecules can be expected during translocation. This was found to be the
case: the acceptor (proflavin) fluorescence decreased and the donor fluo-
rescence increased. The kinetic trace of the proflavin fluorescence was resolved
into three phases with apparent first-order rate constants in the range 1 (fast),
0.1 (intermediate), and The changes in the transfer efficiency
during each kinetic step were calculated from the changes in amplitudes. The
initial (pre-translocative) efficiency was 0.88. It decreased to 0.77, 0.35, and
0.15 during the fast, intermediate, and slow steps, respectively. The fast step
has been assigned to a coordinated displacement of the two tRNA molecules
from their respective sites. Because the transfer efficiency during this phase
was still quite large no significant change of the distance between
the two anticodon loops occurred during the fast step. However, a significant
alteration of the donor–acceptor relationship occurred during the intermediate
kinetic step, which has been ascribed to the release of the deacylated tRNA
molecule. Because translocation as revealed by the puromycin assay was
found to be complete within 60 s after addition of EF-G, the slow kinetic step,
taking several minutes to complete, is unlikely to be a part of translocation.
Paulsen and Wintermeyer(77) further suggested that the E site-bound tRNA
may not be in contact with the mRNA and that the tRNA in the E site may
not be functionally important for fixing the mRNA in the proper position, as
had been previously proposed.(79)
3.9. Distribution of Distances
The end-to-end distance of a flexible polymer chain is characterized by a

distribution among all possible configurations. If one end is labeled with an
energy donor and the other with an acceptor, the observed energy transfer
efficiency must reflect this multiplicity in configuration. Because of the dynamic
nature of macromolecular structure, the observed efficiency of energy transfer

between a donor and an acceptor attached to specific sites within a macro-
molecule or a membranous system can be expected to reflect a distribution in
their separations. Unfortunately, it is not possible to obtain the distribution
from measurements of the steady-state emission intensity of the donor in a
single experiment. A protocol(80) based on theoretical considerations was
proposed in 1971 to obtain the distribution by measurements of steady-state
intensity in a series of experiments in which was varied. It was proposed
to achieve this variation by using different donor–acceptor pairs that were
characterized by different values and were specific for the same donor and
acceptor sites. It was shown that the distribution could be extracted from the
different sets of transfer data by an iterative procedure. In practice, such an
experimental procedure is difficult to apply and has not been used because of
the requirement of many labeled proteins. Since is proportional to donor
quantum yield, it should be possible to vary for a given donor chromo-
phore attached to a macromolecular substrate by using solvent perturbation
in which the donor emission intensity is progressively quenched by addition
of an external quencher molecule such as iodide or acrylamide. This approach
was briefly mentioned(80) and appears considerably less complicated than the
use of different donor–acceptor pairs.
An experimental method based on lifetime measurements was proposed
in 1972 by Grinvald et al.,(81) who outlined various approaches to recover the
distribution in donor–acceptor distances from the monoexponential decay
curve of donor emission. Several model systems(82,83) were used in the inter-
vening years to demonstrate the applicability of this general method, but
progress on its application to proteins has been slow until the past several
years.
3.9.1. Theory
3.9.1.1. Recovery of Distribution from Lifetime Data

If the decay of the donor emission is single exponential, it is given by
where is the donor intensity at zero time, and is the donor lifetime. In
the presence of a single acceptor located at a unique distance R from the
donor, the donor decay is given by
with as the Förster critical distance. The donor still decays monoexpo-
nentially, but with a composite rate constant of corre-
sponding to a lifetime The second term in the
exponent of Eq. (3.28), is the rate constant of energy transfer
to the acceptor.
In a system containing donor–acceptor pairs of different distances, the
donor decay will not be single exponential, but will be dependent upon the
probability distribution of donor–acceptor distances, P(R). Equation (3.28)
becomes
If the donor decay in the absence of the acceptor is described by a sum of i

exponential terms, Eq. (3.29) is modified to
where the are the i donor lifetimes. Thus, direct determination of the donor
decay curve will allow extraction of P(R). Equations (3.29) and (3.30) are
valid for a distribution that remains unchanged during the lifetime of the
donor. Perturbation of the distribution by diffusion may become significant if
the donor lifetime is long and if the donor and acceptor can diffuse relatively
unconstrainedly toward or away from each other.
Fluorescence lifetimes can now be determined with high accuracy by
multifrequency phase fluorometry. In the frequency domain, one measures the
phase shift and the modulation as a function of modulation
frequency The sine and cosine transforms of Eq. (3.30) yield the following
frequency response functions (84,85) :
where is the ith donor lifetime observed in the presence of the acceptor.
If the donor emission in the absence of the acceptor is single exponential, no
summation is needed under the integral sign in Eqs. (3.31) and (3.32).
To evaluate P(R) from time-domain decay data, one selects an
appropriate distribution function P(R) that will yield the best fit between the
calculated according to Eq. (3.29) or (3.30) and the experimental decay
The constants and are obtained from independent experiments.
When frequency-domain data are used, the chosen P(R) is used to calculate
the frequency response function, Eqs. (3.31) and (3.32), for comparison with
experimental and
3.9.1.2. Recovery of Distribution from Steady-State Data

The experimentally determined transfer efficiency (Eqs. 3.13a and 13b) is
conventionally used to calculate a single donor–acceptor separation R by
using Eq. (3.11) once the Förster critical distance is known. If there is a
distribution of distances P(R) for the donor–acceptor pair, the transfer
efficiency is given by (80)
The dependence of the measured transfer efficiency on is different from that

indicated by Eq. (3.11). For each of the donor–acceptor distances, the transfer
efficiency will depend upon the value of used in the experiment. The
problem now is to find different experimental conditions that will yield a
range of values for a given donor–acceptor pair and to recover the
distribution function from the transfer efficiencies measured over the range
of values. Since is proportional to the sixth root of donor quantum
yield (Eq. 3.4), one convenient way to obtain a range of values is through
collisional quenching of the donor quantum yield. This can be accomplished
by using neutral quencher molecules.
The steady-state emission intensities of a donor determined in the absence
and presence of quenching are related to the quencher concentration
[Q] by
where and are the dynamic and static quenching constants, respec-
tively. A plot of versus [Q] should yield a straight line and
allow determination of both and Alternatively, can be determined
from a plot of versus [Q], where and are the donor lifetimes in
the absence and presence of quenching, respectively. The dynamic quenching
constant is then used to obtain the quenched donor quantum yield at
various values of [Q]:
where is the donor quantum yield in the absence of quenching. (A different

symbol is used here for quantum yield to avoid confusion with that for
quencher.) Once a range of values are obtained, they can be used together
with the Förster critical distance determined in the absence of quenching
to calculate the corresponding range of quenched Förster critical distances
It is necessary to use the dynamic quenching constant to calculate the

quenched quantum yield and because the observed steady-state quenching
generally contains both static and dynamic components. The statically
quenched species do not contribute to the emission.
3.9.2. Examples
3.9.2.1. Model System

A model system was recently used by Lakowicz et al.(84) to demonstrate
the feasibility of using the frequency-domain data to recover the distribution
of FRET distances. The molecule N-dansyl undecanoyl tryptamide (TUD)
shown in Figure 3.6 contains a donor (indole) at one end and an acceptor
(dansyl moiety) at the other, connected by a flexible alkyl chain. The decay
and quantum yield of the donor in the absence of the acceptor were deter-
mined independently with N-myristoyl tryptamine (TMA). The frequency
response of the donor emission is shown in Figure 3.7. In the absence of the
acceptor, the donor decay was adequately described by a single exponential

In the presence of the acceptor, the donor decay in TUD clearly
was not single exponential, but could be fitted to a triexponential function.
The donor decay in TUD is expected to remain single exponential if the
donor–acceptor pair is characterized by a single separation. The observed
departure from monoexponential decay is evidence for a range of transfer
efficiencies caused, at least in part, by a distribution in donor–acceptor
distances. The data were then analyzed by using Eqs. (3.31) and (3.32) and a
Gaussian distribution function
where is the standard deviation of the distribution, is the average dis-

tance, and 1, or 2. The half-width (HW) of the distribution (full width
at half-maximum) is The best fit of the data was obtained with
and (Figure 3.8). When HW was fixed at 2 Å, no
satisfactory fit could be obtained. The distributions shown in Figure 3.9 were
recovered from data obtained at several temperatures and using multipliers
1, and 2. The use of a Gaussian distribution must be regarded as an
approximation at this time. It is unclear which multiplier is appropriate
although is appropriate for an infinite flexible chain in three dimensions.
Regardless of which model is used, the recovered distribution is broad and
indicates a wide range of distances.
In a recent study, Gryczynski et al.(85) have demonstrated the validity of
the procedure outlined in Section 3.9.1.2 to recover distance distribution by the
steady-state quenching method. These workers measured values and transfer
efficiencies for TUD and two related peptides in the presence of acrylamide at
seven different concentrations (Table 3.3). As expected, and E decreased
with increasing acrylamide concentration. The distance distributions were
recovered from least-squares analysis of transfer efficiencies. In Figure 3.10 the
first distance distributions recovered from steady-state quenching data for
oligopeptides are compared with the distributions obtained from frequency-
domain lifetime data. The good agreement suggests that the steady-state
method could be a very useful tool for determination of the distribution of
donor–acceptor separations. The uncertainty in the recovered distribution
depends, among other factors, upon the range of values that were used
in the calculations. Although it is generally not possible to obtain a wide

range of values due to the sixth root dependence of on quantum yield,
simulation and experimental data obtained by Gryczynski et al. indicate that
even a 20 % range in values can yield good estimates of the donor–acceptor
distribution. The method is considerably simpler than the previously proposed
method requiring labeling of macromolecules at the same two sites with
several different donor–acceptor pairs and does not require advanced
instrumentation and analysis methods that are essential with the frequency-
domain or time-domain lifetime techniques.
3.9.2.2. Distributions in Proteins

The general procedure used for TUD has been applied to the small protein
TnI (see Figure 3.3). We(86) reinvestigated the distance between Trp-158 and
Cys-133 labeled with IAE by multifrequency phase fluorometry over the range
of 10–300 MHz and extracted the donor–acceptor distance distribution
under different experimental conditions. The analysis of the data was more
complicated than that for TUD because the decay of the single Trp of TnI
was triexponential,(86,87) ranging from below 1 to 6–9 ns. In the presence
of the acceptor, the donor decay was also approximately triexponential, but
the decay was more heterogeneous than in its absence as judged by statistical
criteria. This heterogeneity was taken as manifestation of a distribution in
transfer distances. Shown in Figure 3.11 are the recovered Gaussian distribu-
tions of donor–acceptor distances for native and denaturated TnI. The dis-
tribution for the native protein was characterized by an average distance
of 23 Å, in good agreement with the value previously
reported(43) on the basis of a single donor–acceptor distance and a HW of
12 Å with Upon denaturation increased by about 4 Å, whereas the
HW increased fourfold to 47 Å.
Table 3.4 summarizes additional distance distribution parameters that
were determined for TnI with the complex formed between TnI and TnC.(88)
In the complex the average distance between Trp-158 and Cys-133 was
slightly longer than in isolated TnI, also in agreement with previous
results. However, was not affected by binding to the sites of the
TnC in the complex. This finding differs from previous results based on a
single distance. The reason for the discrepancy is unclear at this time. It is of
interest to note that binding to TnC reduced the half-width of the
distance distribution in Tnl, and the decrease was shown to be statistically
significant. This narrower distribution shows an effect of binding on the
dynamics of TnI, and this effect may be increased dynamic fluctuations. The
altered dynamics may play an important role in the transmission of the
signal from TnC via TnI to activate actomyosin ATPase.
Simulation studies and examination of the ratio surfaces for the
simulated frequency-domain data indicated that values of and HW can be
recovered to within 2 Å.(88) Probe mobility and the uncertainty in the orienta-
tion factor can contribute to the observed distribution width. These
problems were considered in detail, and the possible effect of on HW was
investigated by measurements of anisotropy decay.(85,88) While it was not yet
possible to assign unequivocally the various factors contributing to any
observed distribution width, it was concluded that experimental half-widths in
excess of 10 Å, such as those observed with TnI in the native state, must
reflect to a certain extent local dynamic fluctuations of the protein. The dis-
tribution was used to follow structural changes as a function of denaturation
by guanidine hydrochloride (GuHCl). The half-width increased progressively
and dramatically with increasing denaturant concentration from about 12 Å
to a final level of about 50 Å, which was observed at The
midpoint of the transition was near 1.5 M GuHCl. The change in followed
a similar pattern. TnI is known to be stabilized against denaturation by
complexation with TnC in the presence of This protection was also
evident from the recovered distributions determined under these conditions.
The large HW observed in the presence of GuHCl is compatible with the
expectation of a wide range of distances and decreased dynamic fluctuations
in the denatured protein. These results demonstrate that the distributions of
FRET distances in protein can be recovered with good accuracy and are use-
ful in studies of protein folding and unfolding.
The steady-state quenching method was also used to determine the

distance distribution of TnI in a manner similar to that described in
Section 3.9.2.1 for model systems. The recovered distributions (85,88) are in
excellent agreement with those shown in Figure 3.11. The good agreement
provides further confidence in the steady-state method.
We recently recovered the distribution of the distances between Met-25
and Cys-98 in TnC. Of interest is the reduction of the mean distance and the
half–width of the distribution induced by calcium binding to the two calcium-
specific sites. These decreases are observed with both isolated TnC and TnC
complexed to TnI(89). The results from this study and those on the distance
distributions of TnI in the TnI–TnC complex indicate that the binding of
activator calcium to its receptor sites (TnC) in muscle alters the conforma-
tional dynamics of both proteins. The alternate interaction of TnI with actin
and TnC during cycles of relaxation–contraction may be closely related to the
changes in dynamic fluctuations in these proteins, and these fluctuations may
provide a mechanism by which the calcium signal is transferred from its
receptor via TnI to distant sites on the actin filament.
A fourth example of distance distribution is the distances of
myosin S-l (distance 6, Fig. 2). The distribution parameters recovered under
several experimental conditions are listed in Table 3.5.(90) The previously
observed decrease in induced by MgADP was reproduced from the
recovered distribution. In contrast to TnI, denaturation of S-l in 6 M
guanidine hydrochloride resulted in a small increase in the half-width and a
decrease of only 3 Å in the mean distance. The lack of a significant denatura-
tion effect on the distribution half-width indicates that the polypeptide segment
is question has very similar conformational dynamics regardless of whether
the protein is native or denatured. The conformation of the segment
in native S-l approaches a random coil. This is the first direct physical
evidence for an extreme segmental flexibility in this region of the S-l heavy
chain. It is not possible to obtain this information from other types of
experiments.
Recovery of the distribution of FRET distances in proteins from time-
domain data has also been accomplished in several studies. Amir and Haas(91)
determined the distribution of several intramolecular distances in bovine
pancreatic trypsin inhibitor (BPTI). They labeled the N-terminal amino
group with the energy donor (2-methoxy-l-naphthyl)methyl (MNA) and the
group of one of the four lysine residues (positions 15, 26, 41, and 46)
with the acceptor [7-(dimethylamino)coumarin-4-yl]acetyl (DA-coum). For
each of the four derivatives with attached donor and acceptor, the
monoexponential decay curve of the donor emission was used to
recover the distance distribution. The half-widths of these distributions were
in the range 9–11 Å, and the average distances ranged from 21 to 34 Å. When
the sizes of the donor and acceptor probes were estimated and taken into
account, the recovered average distances were very close to those expected
from the crystal structure of BPTI.
In another study of the distribution of distances based on time-domain
data, Haas et al.(92) labeled Glu-49 and Asp-53 of bovine pancreatic RNase
with ENA (ethylenediamine monoamide of 2-naphthoxyacetic acid) as energy
donor and the N-terminal amino group with l-fluoro-2,4-dinitrobenzene as
energy acceptor. The attached ENA decayed monoexponentially in the
absence of the acceptor. The distribution of distances recoverd from data
obtained in 50% glycerol showed an average distance of and a half-
width of The average distance compares favorably with the value
previously determined in aqueous solution on the basis of a single donor–
acceptor separation.(68) Upon denaturation by 6 M guanidine hydrochloride
in 26% glycerol the average distance increased to and the half-width
to These authors also discussed in detail possible contributions
to the observed half-width. Since the attached donor probe used in this study
has a considerable length (eight or nine single bonds and two amide bonds),
probe flexibility (as opposed to protein dynamics) may have contributed
significantly to the observed distribution recovered for the protein in the
native state. Nevertheless, these results qualitatively corroborate those we
have obtained with TnI in that the parameters of the distribution of FRET
distances are sensitive to transition from an ordered state to a partially dis-
ordered or completely disordered state.
In two recent reports Brand and co-workers reported the distributions of
the distances between two specific residues of a staphylococcal nuclease
mutant that were recovered from time-domain data as a function of tem-
perature(93) and guanidine hydrochloride concentration.(94) Bimodal distribu-
tions were needed to fit the data obtained at high temperature or in high
guanidinium concentration, providing evidence for two denatured states of the
mutant protein consistent with a circular dichroism study (95) that
staphylococcal nuclease mutants can exist in two denatured states. These
results clearly suggest potential applications of the distribution of FRET
distances to protein folding/unfolding problems.
In closing, mention should be made of the possibility of extracting

distance distributions from measurements of the fluctuation of either donor or
acceptor steady-state fluorescence intensity. Haas and Steinberg(96) presented
a very interesting theoretical treatment in which the autocorrelation function
of the fluorescence intensity is derived for a flexible polymer chain in terms of
(1) the diffusion coefficient of one set of chain ends relative to the other and
(2) the equilibrium distribution function of distances between donor and
acceptor chromophores. Simulations were carried out for a series of oligomer
chain molecules which were labeled with an energy donor at one end and an
energy acceptor at the other. The equilibrium distance distributions of these
molecules had been previously obtained from the nonexponential decay curve
of the donor emission.(82) The results showed that the intensity fluctuation
can be large enough to be experimentally useful in the study of molecular
dynamics of macromolecules. In spite of the simplicity of the derived
autocorrelation function, no experimental studies have been reported because
of the enormous instrumental problems associated with such fluctuation
measurements.
3.10. Summary and Prospects
The foundation for FRET studies of biological systems is well estab-

lished. For simple macromolecules the determination of energy transfer by
measurements of donor steady-state intensity is relatively simple to carry out,
but for macromolecular assemblies the determination is more complicated.
Lifetime measurements for energy transfer should offer advantages over the
steady-state method because they are independent of concentrations, but they
are considerably more difficult to perform. The accuracy of the measured
donor–acceptor distances are frequently limited to the uncertainty of the
orientation factor, except for special cases where isotropic oscillators (e.g.,
lanthanide ions) are used as the donor and acceptor. However, by using
donor–acceptor pairs that meet certain spectroscopic criteria and by also
performing ancillary experiments on fluorescence depolarization, one can
place an estimate on the error in distance resulting from the uncertainty in
In many reported studies, the error in assuming is not more than 30%.
The general method has already provided answers to a number of
questions in certain biological systems. Among these is the demonstration of
global structural perturbation resulting from a biological event. Individual
donor–acceptor distances within a macromolecule or in an assembly of such
molecules can be useful in providing an approximate molecular shape of the
system in solution. Most donor–acceptor pairs are useful in the distance range
10–60 Å, which is the range of molecular dimensions for many globular
proteins. The large FRET distances are complementary to the considerably

smaller distances that are determined by NMR for construction of solution
molecular structure.
Recent applications of FRET include stopped-flow studies of enzyme
catalysis and the kinetics of changes in energy transfer that occur during a
biological event. The time constants of these changes can provide an insight
into the biological relevance of global structural perturbations detected by
FRET.
The recent success in recovering distributions in donor–acceptor distances
by both pulse and phase fluorometry has opened a new direction for experi-
mental investigation of intramolecular dynamics. The feasibility of using
steady-state emission intensity data to determine distance distributions in
peptides and proteins has now been experimentally demonstrated. Because
of the relative ease with which such data can be generated, the steady-state
quenching method should be of widespread usefulness in studies of macro-
molecular dynamics. The experimental distribution may serve to test the
validity of distance distributions predicted from theoretical considerations.
Because the distance distribution is determined by the overall dynamics of the
macromolecule, it should be useful in studies of the mechanism of protein
folding and elucidation of the sequence of formation of its intermediates along
the folding pathway. The protocols for recovering distance distributions by
multifrequency fluorometry have been extended to cases where the decay of
the donor emission is multiexponential, but are still limited to the situation
where there is a single acceptor for each donor. When the algorithms are
extended to multiple acceptors, it should be possible to determine clustering
of lipids or proteins in membranes and ion distribution around polyelectro-
lytes such as nucleic acids.
Acknowledgment
The preparation of this chapter and the research carried out in my

laboratory and described herein were supported in part by NIH grants AR
31239 and AR 25193.
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4
Least-Squares Analysis of
Fluorescence Data
Martin Straume, Susan G. Frasier-Cadoret,
and Michael L. Johnson
4.1. Introduction
The evaluation of mechanistic parameters, such as fluorescence lifetimes

and amplitudes, by the application of nonlinear least-squares methods for the
analysis of experimental data is a mathematically ill-posed problem. In effect,
this means that it is much more difficult to derive fluorescence lifetimes from
experimental data than it is to calculate synthetic data from known values
of the fluorescence lifetimes and relative amplitudes. The reader should be
aware that nonlinear least-squares parameter estimation techniques are not
guaranteed to find a set of mechanistic parameters which will provide a good
description of any particular set of experimental data. Furthermore, even if
a set of mechanistic parameters is found to fit the experimental data within
reasonable statistical precision, there still is no guarantee that those parameters
will be the unique, or the best, set of answers. However, when the assumptions
which govern the parameter estimation techniques are carefully obeyed,
experimental data can be analyzed to yield useful information about an
experimental system.
Nonlinear least-squares analysis is a numerical procedure for estimating
a set of parameters, of an equation, such that this equation will
describe a particular set of data points, and These procedures work by
successive approximation; that is, given an estimate of the values of the
nonlinear least-squares algorithm returns a more accurate approximation of
Martin Straume, Susan G. Frasier-Cadoret, and Michael L. Johnson • Departments

of Pharmacology and Internal Medicine, University of Virginia Health Sciences Center,
Charlottesville, Virginia 22908. Present address for M.S.: Department of Biology, The Johns
Hopkins University, Baltimore, Maryland 21218.
Topics in Fluorescence Speciroscopy, Volume 2: Principles, edited by Joseph R. Lakowicz. Plenum
177
178 Martin Straume et al.
This approximation procedure is then applied iteratively until the values of

the parameters being estimated, do not change within some specified limit.
Linear least-squares analysis is a special case of the more general non-
linear least-squares procedures which requires only a single iteration to
determine correct values for the parameters. A system of equations is linear
if all of the second and higher order derivatives of the function with
respect to all of the individual parameters which are being estimated, are
equal to zero. Examples of linear equations are straight line and quadratic
equations. Because the only significant difference between linear and nonlinear
least-squares techniques is the number of iterations required to reach a stable
solution, we will only discuss the more general nonlinear least-squares
parameter estimation procedures in this chapter.
All linear and nonlinear least-squares parameter estimation techniques
are based on a series of assumptions. Only when these assumptions are strictly
obeyed can the use of least-squares parameter estimation techniques be
justified or the results obtained be considered valid. When these assumptions
are not obeyed, the least-squares techniques are, at best, approximate and
their applications somewhat empirical. Nevertheless, least-squares techniques
have proven to be extremely useful for the analysis of data from fluorescence
experiments.
The collection of experimental data and the analysis of these data by a
technique such as nonlinear least squares is not a process of two independent
sequential steps. The method of choice for the analysis of the experimental
data is dependent on the inherent random and systematic uncertainties and
experimental limitations of the actual experimental data. Consequently, the
data need to be collected in such a manner that the concomitant experimental
uncertainties of the data are compatible with the assumptions inherent to the
method of analysis.
The purposes of this chapter are, first, to review the assumptions upon
which nonlinear least-squares parameter estimation techniques are based
and discuss the consequences of these assumptions; second, to describe the
mathematical details of some of the nonlinear least-squares techniques for
parameter estimation, confidence interval evaluation, and confidence interval
propagation, and, third, to review other measures of goodness of fit with
particular reference to the information which can be obtained from the dif-
ferences betwen the actual experimental data and the “maximum likelihood”
fitted function. We discuss only those techniques which we have found to be
particularly useful and do not attempt to review all of the nonlinear least-
squares methodologies in the literature. We will also provide a series of specific
examples of the application of these techniques to the analysis of fluorescence
data.
The primary emphasis of this chapter will be on the analysis of
fluorescence lifetime data. However, the techniques and methodologies of least-
Least-Squares Analysis of Fluorescence Data 179
squares analysis were developed for, and are commonly applied to, a wide
range of types of experimental data in fields such as physics, chemistry,
biology, sociology, and economics.
4.2. Basic Terminology
Before we proceed with the mathematical basis of the assumptions

and methodologies of nonlinear least-squares analysis, terms such as data
sets, dependent variables, independent variables, fitting functions, and the
parameters of a fitting function must be defined. In each case examples are
presented to clarify these concepts.
For time-domain fluorescence lifetime measurements a data set consists
of a series of observations of fluorescence intensity and lamp intensity as a
function of time. For frequency-domain fluorescence lifetime measurements a
data set consists of a series of observations of amplitude modulation and
phase shift of the emitted light as a function of the modulation frequency of
the excitation light. A typical data set for steady-state fluorescence intensity is
a series of observations of fluorescence intensity as a function of emission
wavelength. For the mathematical discussions we refer to a data set as a series
of observations, and where the subscript refers to an individual data
point within the data set.
Independent variables are those quantities whose values we can control
by appropriate settings of the instrumentation or sample preparation. For
example, time is the independent variable for time-domain fluorescence
lifetime measurements, frequency is the independent variable for frequency-
domain fluorescence lifetime measurements, and wavelength is the independent
variable in the steady-state intensity example above. We refer to independent
variables as
Dependent variables are those quantities whose values are actually
measured by the experimental protocol. For example, fluorescence intensity is
the dependent variable for time-domain fluorescence lifetime measurements,
amplitude modulation and phase shift are the dependent variables for
frequency-domain fluorescence lifetime measurements, and intensity is the
dependent variable in the steady-state fluorescence example. We refer to
dependent variables as
For convenience in the discussion of the numerical methods, we present
the independent and dependent variables in two dimensions as a series of
scalar quantities, and However, all of the mathematical procedures
apply equally well when and are vectors in multiple dimensions. For
example, for frequency-domain fluorescence lifetime measurements there
are actually two dependent variables, amplitude modulation and phase shift.
A two-dependent-variable problem such as this can easily be handled by
considering each of the as a vector of length two, the first element being
the amplitude modulation of the emitted light and the second being the phase
shift of the emitted light.
The mathematical relationship between the independent and dependent
variables is referred to as the fitting function:
where α is a variable-length vector of parameters of the fitting function,

The difference between the individual data points, and the fitting
function evaluated at the "optimal" values of is expressed in Eq. (4.1) as
These differences are sometimes referred to as
the residuals of the fit.
In fluorescence lifetime measurements the simplest representation of the
fluorescence intensity, as a function of time, t, is given by
where is the fluorescence amplitude at time zero, and is the fluorescence

lifetime. For time-domain experiments the corresponding fitting function,
, is a convolution integral of the intensity decay, and the lamp
intensity, L(t):
where t is the variable of integration over time from 0 to the vector of

fitting parameters, consists of and and corresponds to the time
value of a particular data point. (1) The lamp intensity as a function of time,
L(t), is experimentally determined for each experiment.
Since frequency-domain experiments have two dependent variables,
amplitude modulation and phase shift, there are actually two fitting functions,
and where the “am” and “ps” subscripts refer to either
amplitude modulation or phase shift (1) :
and
where is equal to the excitation modulation frequency, and

For frequency-domain experiments where is given by Eq. (4.2), the

vector of fitting parameters, has only one element, the fluorescence life-
time, The total amplitude, has been canceled by normalization of the
integrals [the denominator of Eqs. (4.6) and (4.7)].
It should be noted that when the intensity decay law consists of multiple
exponentials, or some nonexponential form, Eqs. (4.3)–(4.7) are still valid,
and the fitting parameters, are the parameters characteristic of a different
intensity decay law, Some examples of more complex decay laws are
presented later in this chapter.
4.3. Assumptions of Least-Squares Analysis
The desired result of the analysis of any set of data, and is to

evaluate a set of parameters, of a fitting function, The analysis
process should yield the parameter values that have the maximum likelihood
of being correct. Furthermore, the function evaluated at these optimal
parameter values must be a reasonable description of the actual experimental
data in the absence of experimental uncertainties. In order to correctly obtain
the maximum-likelihood parameter values, the function and the
distribution of experimental uncertainties must satisfy a series of assumptions.
These assumptions are presented here, and their effects on the analysis process
are discussed.
All nonlinear least-squares parameter estimation procedures have six
inherent basic assumptions. These are:
1. All of the experimental uncertainties of the data must be attributable
to the dependent variables,
2. The experimental uncertainties of the dependent variables, must
follow a Gaussian (normal or bell-shaped) distribution.
3. No systematic uncertainties can exist in either the dependent, or
independent, variables.
4. is the correct mathematical description of the data.
5. There must be a sufficient number of data points to yield a good
random sampling of the parent population of residuals.
6. The data points must be independent observations.
The first assumption means that the values of the independent variables
are known to much greater precision than the values of the dependent
variables, so that the parameter estimation algorithm can ignore any
experimental uncertainties in the independent variables. For a two-dimensional
Y versus X analysis the least-squares parameter estimation algorithm will
minimize the sum of the squares of the vertical (Y axis or ordinate) deviations,
as shown in Figure 4.1. It is particularly important to realize that it is not
the perpendicular or horizontal distance from each data point to the best-fit
curve which is minimized. Therefore, in order for a least-squares analysis to
correctly predict the maximum-likelihood parameter values, the analytical
problem must be formulated, and the data collected, such that the X axis
(abscissa) is known to much greater precision than the Y axis (ordinate).
Experimental uncertainties in the independent variables cannot be correctly
compensated for by “appropriate weighting factors.” In general, there is no
way to circumvent this requirement and still correctly utilize a least-squares
procedure. A different method of analysis which does allow Gaussian
distributed experimental uncertainty on both the dependent and independent
variables is presented elsewhere. (2)
The first assumption allows transformations of the independent variables,
the X axis or abscissa. For example, in frequency-domain fluorescence lifetime
experiments the independent variable is frequency, but it is usually more
convenient to graphically represent the experimental data as a function of the
logarithm of frequency. Such a logarithmic transformation of the independent
variables does not invalidate the least-squares procedure.
The data obtained from fluorescence experiments are usually consistent
with the first assumption. For time-domain fluorescence lifetime measurements
the uncertainty in the time measurement (the X axis or abscissa) is small.
For frequency-domain fluorescence lifetime measurements the modulation
frequency of the excitation light (the X axis or abscissa) is known to excellent
presicion. The precision of the wavelength (the X axis or abscissa) for the
steady-state fluorescence case is also sufficient for the least-squares method of
analysis.
The second assumption states that experimental uncertainty of the
dependent variables, must follow a Gaussian distribution. This means
that if a particular data point was measured an infinite number of times,
a frequency histogram of the observed values for that point would follow
a Gaussian distribution. Figure 4.2A depicts a distribution of this form.
This assumption is met by many, but not all, experimental procedures.
In particular, the validity of this assumption, and the consequences of
violating this assumption, have been discussed with reference to the use of
the method of moments for the analysis of time-domain fluorescence lifetime
measurements.(3)
Nonlinear transformations of the dependent variables, the Y axis or
ordinate, violate the second assumption. While Figure 4.2A shows a Gaussian
frequency distribution of a dependent variable, Figure 4.2B illustrates the
perturbation of the same frequency distribution introduced by performing a
logarithmic transformation on the dependent variable. It is clear that the
frequency distribution in Figure 4.2B is not Gaussian and thus violates the
second assumption of least-squares parameter estimation. For example, in
time-domain fluorescence lifetime experiments the counting uncertainty in the
dependent variable, the frequency of counts within a given time range, follows
a Poisson distribution. When the frequency of counts is large, these Poisson
distributions can be approximated by a Gaussian distribution with a standard
deviation equal to the square root of the mean. Consequently, the second
assumption is usually reasonable for time-domain fluorescence experiments
when the data are represented as counts as a function of time. A common
method employed to analyze this type of data, which violates this assumption
of least-squares analysis is to plot the logarithm of the number of counts
as a function of time. For the case of a single exponential decay this plot
should be a straight line. However, such a logarithmic transformation of
the dependent variables produces a skewed distribution of experimental
uncertainties which is no longer a Poisson distribution and cannot be
approximated by a Gaussian distribution, as shown in Figure 4.2B, and thus
contradicts the assumptions of the least-squares procedure. Consequently, for
this example, it is not valid to use any least-squares procedure to determine
the slope of a plot of the logarithm of counts as a function of time.
The third assumption states that the experimental data have no systematic
uncertainties. All parameter estimation procedures make this assumption.
Systematic errors are a problem that must be corrected in the experimental
protocol rather than by the analysis procedure. The only way to circumvent
this assumption in the analysis procedure is to include a series of terms into
the fitting function, to describe all of the systematic uncertainties. The
vector of fitting parameters, will then include parameters which describe the
systematic uncertainties. A possible systematic uncertainty for time-domain
fluorescence lifetime measurements might be a slight difference in the initial
time value between the lamp intensity profile, L(t), and the observed fluo-
rescence intensities, If such a difference exists, and it cannot be removed
by instrument calibration, then a time offset term, could be included in
Eq. (4.3) such that
where the vector of fitting parameters, now includes and as in

Eq. (4.2), and the time offset term, The least-squares estimation procedure
then involves evaluation of the three parameters implicit in Eq. (4.8) rather
than the two parameters of Eq. (4.3).
The fourth assumption states that the choice of the fitting function,
is the correct mathematical description of the phenomenon being
observed. This seems obvious, but an example is worth noting. Consider the
case of fluorescence lifetime measurements of a solution containing a random
distribution of fluorescence donors and fluorescence acceptors such that
energy transfer occurs. In this case the form of the fluorescence intensity as a
function of time, can sometimes be approximated by (4)
where b is proportional to the concentration of the fluorescence acceptor and

an encounter radius. Analysis of data of this type could also be performed by
assuming that the intensity decay law is the sum of a series of k exponential
decays of the form
where the vector of fitting parameters, consists of a series of amplitudes,

and lifetimes, If k is sufficiently large, then
Eq. (4.10) is capable of describing the intensity decay to any desired precision.
However, in that case, the values of and will have
little, or no, actual physical meaning. However, if the data were analyzed by
Eq. (4.9), then the resulting parameters would be physically meaningful.
It is a necessary, but not a sufficient, condition for a physically meaningful
analysis of experimental data that the functional form, be capable of
describing the experimental data to within the precision of the data.
The fifth assumption requires that the number of data points be sufficient
to ensure a good statistical sampling of the random experimental uncertainties.
The exact number of data points required to satisfy this assumption is difficult
to specify and differs for each set of experimental conditions. Most time-
domain fluorescence lifetime instruments collect a minimum of several hundred
data points, which is usually sufficient for this type of experiment. However,
some of the older frequency-domain fluorescence lifetime instruments do
not produce an adequate number of data points for a proper least-squares
analysis since they collect data at only three fixed modulation frequencies.
The newer multi-frequency fluorometers can collect data at any number of
frequencies and can thus provide ample data for the analysis.
The last assumption states that each experimental observation,
is independent of any other data point. This means that the concomitant
experimental uncertainties in a particular data point do not influence the
experimental uncertainties in any other data point. For example, if the multi-
channel analyzer used for time-domain fluorescence lifetime experiments did
not provide stable time windows, then the uncertainties in the data points
would appear to be correlated with each other; that is, too many counts in
one channel would probably be correlated with too few counts in an adjacent
channel. Error dependence between data points might cause the analysis
procedure to produce erroneous parameter values.
Utilizing these six basic assumptions, it is relatively easy to demonstrate
that the method of least squares does produce values for the parameters,
with the highest probability, that is, maximum likelihood, of being correct. If
the parameters, are correct and all six assumptions are met, we can write
the probability of observing a particular dependent variable, at a
particular independent variable, as being equal to
where is the standard deviation of the random experimental uncertainty at

a particular data point, and Z is a proportionality constant. Equation (4.11)
also gives the probability, that the values of the fitting parameters,
are correct for a data point, and
The total probability that the values of the fitting parameters, are correct
for a complete data set is the product of the probabilities for the individual
data points:
where the product, and summation, apply to each of the n data points
with subscript i. The maximum probability that the fitting parameters are
correct, will occur when the summation in the exponential of Eq. (4.13)
is a minimum. Consequently, a procedure which will minimize this summation
will yield the set of fitting parameters which have the maximum likelihood of
being correct. We can thus define a NORM of the data to be minimized to
yield the maximum-likelihood parameter values as
where NDF is the number of degrees of freedom. NDF is defined to be the

number of independent experimental observations minus the number of
constraints. Typically, NDF is evaluated as the number of data points times
the number of dependent variables per data points minus the number of
fitting parameters. Equation (4.14) is the standard weighted least-squares
NORM of the experimental data points, which is commonly written as the
sample variance, Consequently, with the six assumptions stated above, the
parameter values, with the maximum likelihood of being correct will be
given by the method of least squares.
It is possible, of course, that a minimum may not exist for the least-
squares NORM, s2, and consequently the values of the fitting parameters
cannot be determined by any least-squares method. It is also possible that
more than one minimum of the least-squares NORM exists, in which case the
values of the fitting parameters will not be unique.
If any of the assumptions are not rigorously met, then the least-squares
method may yield a set of parameter values which do not have the maximum
likelihood of being correct. It is impossible to state a priori how much of an
error in the estimated parameters will be introduced if one, or more, of the
assumptions is not correct. Therefore, it is important to try to abide by the
assumptions if a least-squares method is to be used for the analysis of
experimental data. Least-squares methods are commonly utilized for the
analysis of fluorescence data because, in most cases, the assumptions can be
reasonably applied.
4.4. Least-Squares Parameter Estimation Procedures
The nonlinear least-squares minimization process can be performed by a

number of numerical procedures. All of the methods will yield equivalent
results for the majority of problems. Consequently, we will only present two
of the methods, a modified Gauss–Newton procedure(5) and the Nelder–Mead
simplex procedure,(6) which we have found to be particularly useful. We will

also discuss the advantages and disadvantages of each of these methods.
The reader is reminded that although the algorithms will be presented in
terms of a two-dimensional problem, Y versus X, they are easily expanded to
more dimensions. This expansion allows the analysis of experimental data with
multiple dependent variables, such as amplitude modulation and phase shift for
frequency-domain fluorescence lifetime measurements. This scalar-to-vector
expansion also allows for the analysis of experimental data with multiple
independent variables, for examples, steady-state intensity measurements as a
function of emission wavelength, temperature, quencher concentration, etc.
4.4.1. Modified Gauss–Newton Algorithm
The Gauss–Newton nonlinear least-squares algorithm is a procedure that

starts with approximate values for the fitting parameters, and generates a
better estimate of the fitting parameters. The algorithm is applied iteratively
until the values of the fitting parameters do not change within a specified
precision. Once the fitting parameters are stable, that is, do not change within
a specified tolerance, they will correspond to a minimum of the least-squares
NORM, (Eq.4.14).
In the Gauss–Newton algorithm the data points are assumed to be
approximated by the fitting function evaluated at the values of the fitting
parameters after iterations:
The fitting function evaluated after iterations is expressed as a first-order

series expansion of the function evaluated after k iterations:
where the i subscripts refer to a particular data point, the j subscripts refer to
a particular fitting parameter, and the k and superscripts refer to the
iteration number. Equation (4.16) is actually a series of equations, one for
each data point. This series of equations can be used to evaluate the values
of the fitting parameters for the iteration, because the form of the
fitting function, is known and we know the values of the fitting
parameters from the previous iteration, For the first iteration we must
arbitrarily assign the initial values of the fitting parameters, The procedure
is repeated until the fitting parameters, do not change significantly for
several iterations.
It should be noted that the only difference between linear and nonlinear
least-squares analysis is the number of significant terms in Eq. (4.16). If all the
second and higher order derivatives of the function with respect to the
Fitting parameters are zero, then the first-order series expansion, Eq. (4.16), is
exact. If Eq. (4.16) is exact, then the solution for the fitting parameters,
will be exact, and the process requires only a single iteration to reach stable
values of the fitting parameters, If the second and higher derivatives are not
zero, then Eq. (4.16) is only approximate, the parameter values produced will
be only approximately correct, and the process will require multiple iterations
before stable parameter values are attained.
Some authors have claimed that linear least-squares analysis does not
require an initial estimate, or guess, of the values of the fitting parameters,
This is not strictly correct. All of the standard equations for the solution of
linear least-squares problems can be derived from the Gauss-Newton non-
linear least-squares procedure by a careful selection of the initial values of the
fitting parameters and the use of a single iteration. When zero is used as the
initial value for the fitting parameters, then all terms which are explicitly
multiplied by the fitting parameters become zero and can be eliminated. So,
although it seems that no initial estimate of the parameter values is required
for the linear case of least-squares analysis, actually the initial estimate has
been assumed to be zero.
The evaluation of the fitting parameters at the next iteration, is
most easily explained in matrix notation such that Eq. (4.16) can be written
as
where is a vector of weighted residuals whose individual elements are
P is a matrix of weighted partial derivatives whose elements are
and is a correction vector whose elements are
A weighting factor, has been included in Eqs. (4.18) and (4.19) to

allow for a variation in the magnitude of the random experimental uncertain-
ties of the individual data points. Here, represents the standard deviation
of the experimental uncertainty in the dependent variable and has the same
form and meaning as the weighting factor described in the least-squares
NORM (Eqs. 4.11, 4.13, and 4.14). If the value of the dependent variable,
represents the mean of a group of observations, then is the standard error
of the mean (SEM) of that group of observations, not the standard deviation
(SD) of the group of observations.
The matrix form of the series expansion, Eq. (4.17), can be solved for the
correction vector, if both sides of Eq. (4.17) are multiplied by the transpose,
of the partial derivative matrix, P, as in Eq. (4.21). Standard matrix
inversion techniques can then be applied to evaluate the correction vector,
as in Eq. (4.22):
Equation (4.17) can also be solved directly by singular-value decomposition

techniques. The singular-value decomposition method for the solution of
systems of linear equations can be implemented with the FORTRAN subroutine
SVD (Ref. 7, p. 192) or the EISPACK † FORTRAN subroutines SVD and MINFIT.(8, 9)
Once the correction vector has been evaluated, then the values of the fitting
parameters for the next iteration, can be evaluated:
The Gauss–Newton process, Eqs. (4.15)–(4.23), is repeated until the values

of the fitting parameters, do not change with successive
iterations to within some specified precision.
In the Gauss-Newton procedure the magnitude of the correction vector,
after iterations is proportional to the square of the magnitude of the
correction vector after k iterations. This is referred to as a “quadratic
convergence.” This means that once the parameter values, are close to the
maximum-likelihood values, convergence is extremely rapid. Table 4.1 shows
the Gauss-Newton procedure used to evaluate the square root of nine. In this
example, the correction vector, decreases by a factor of 2.5 for the first
iteration, by a factor of 4.25 for the second iteration, by a factor of 16 for
the third iteration, by a factor of 256 for the fourth iteration, and by more
than 18,000 for the last iteration. It is interesting to note that because of this
quadratic convergence property most computer systems, and hand calculators,
use the Gauss-Newton procedure to evaluate square roots.
Several different criteria are commonly used to decide if the Gauss–
Newton procedure has converged. For example, it is commonly assumed that
if the least-squares NORM, that is, the sample variance does not change
†
The EISPACK software package is a library of FORTRAN subroutines to perform eigensystem
manipulations. It is available from the International Mathematical and Statistical Library
(IMSL) Distribution Service, P.O. Box 4605, Houston, Texas 77210-4605, for a nominal charge.
by one part per thousand then the process has converged. A much better
criterion is that both the values of the fitting parameters, and the variance
change by less than one part per hundred thousand between successive
iterations. It is realistic to apply this more stringent criterion to the Gauss–
Newton procedure because usually only a few additional iterations are required
to produce the more rigorous convergence.
The derivation of the Gauss–Newton procedure presented in Eqs.
(4.15)–(4.23) has not been based on the minimization of the least-squares
NORM (i.e., sample variance ) in Eq. (4.14). However, from a careful
examination of Eq. (4.21), it can be demonstrated that the Gauss–Newton
procedure is a least-squares minimization procedure. It can be shown that the
elements of the vector on the left-hand side of Eq. (4.21), are propor-
tional to the derivative of the least-squares NORM, with respect to the
corresponding fitting parameter, The Gauss–Newton process is repeated
until the correction vector, on the right-hand side of Eq. (4.21) is equal to
zero. Thus, at convergence the terms on the left-hand side of Eq. (4.21), the
elements of the vector, are proportional to the respective first derivatives
of the least-squares NORM, and the right-hand side of Eq. (4.21), is
equal to zero. The standard method to locate a minimum of any function,
such as the least-squares NORM, is to set the first derivative of the function
equal to zero and to solve for the variable parameters, The Gauss–Newton
procedure is, consequently, a least-squares minimization procedure since the
first derivatives of the function are equal to zero at convergence.
For typical parameter estimation problems to which the Gauss–Newton
procedure is applied, the matrix (P'P) in Eq. (4.21) is difficult to invert
because it is almost singular. Consequently, the numerical procedure is very
sensitive to numerical truncation and round-off errors. For example, a typical
set of fitting parameters for time-domain fluorescence lifetime experiments is
composed of an amplitude of tens of thousands of counts per time channel
and a relaxation time of five nanoseconds. If the fitting function is written

in units of counts and seconds, the magnitude of the fitting parameters will
differ by 13 orders of magnitude, and consequently the elements of the (P´P)
matrix could vary by approximately 26 orders of magnitude. Single-precision
computer computations are accurate to about five orders of magnitude, and
double precision is accurate to approximately 12 orders of magnitude. There-
fore, it is important to scale the fitting parameters so that the individual
elements of the (P´P) matrix are of comparable size within the precision of the
computer calculations. For the time-domain fluorescence example, a better
choice of units is percentage of full-scale counts and nanoseconds. All of the
calculations should be performed in double precision, not just the matrix
inversion. It is also important to choose a computer algorithm for the solution
of Eq. (4.21) which is proven to work with near-singular matrices. The Square
Root Method(10) and the LINPACK FORTRAN subroutines DPOSL and DPOFA(11)
work well for near-singular matrices. The singular-value decomposition
method for the solution of systems of linear equations also works well under
these conditions (see Ref. 7, p. 192, and Refs. 8 and 9).
The advantage of the Gauss–Newton procedure is that with reasonable
initial values of the fitting parameters it will usually converge very rapidly.
The disadvantages are that it does not always converge, that it requires
derivatives of the function with respect to the fitting parameters, and that it
can only be used for a least-squares minimization process. The failure to con-
verge is a consequence of neglecting the higher order derivatives in Eq. (4.16)
and thus is a problem which might occur with any nonlinear problem. This
problem is minimized if the initial values of the fitting parameters, are close
to the maximum-likelihood answers. Only one of the numerous methods to
improve the convergence properties of the Gauss–Newton algorithm will be
discussed here.
One simple way to guarantee convergence of the Gauss–Newton algo-
rithm is a small modification of Eq. (4.23). If the value of the least-squares
NORM, the sample variance evaluated at the estimated fitting parameters
for the next iteration, is greater than or equal to the value of the sample
variance evaluated for the current iteration, then each element of the
correction vector, is divided by two and a new set of values is calculated for
the next iteration, Otherwise, the analysis continues using the standard
Gauss–Newton procedure as outlined above. This cycle may be repeated
several times for each iteration of the Gauss–Newton procedure to ensure that
the sample variance for the next iteration is always less than the sample
variance for the current iteration.
The LINPACK software package is a library of FORTRAN subroutines to perform linear algebra
manipulations. It is available from the International Mathematical and Statistical Library
(IMSL) Distribution Service, P.O. Box 4605, Houston, Texas 77210-4605, for a nominal charge.
The Gauss–Newton algorithm uses the first derivatives of the fitting

function, with respect to each of the fitting parameters, This
implies that first derivatives of the function exist, that the first derivatives are
continuous, and that the second derivatives are continuous so that the first
derivatives vary smoothly. However, the algorithm needs only a numerical
value for the derivative of the fitting function evaluated at the current fitting
parameters, and the values of the independent variables, The algorithm
does not use, or need, explicit equations for the derivatives. It is therefore
recommended that the values of the derivatives be evaluated by a three- or
five-point Lagrange differentiation formula. (12) The five-point formula for the
derivative, of a function f(z) evaluated at z is
where is a small numerical increment of z. The value of should be chosen

such that
The error in the evaluation of the derivative is proportional to the fourth

power of the increment, and to the fifth derivative of the function
evaluated at where is in the range
Once convergence has been reached the information matrix, (P´P) in
Eqs. (4.21) and (4.22), contains some useful information about the parameter
estimation process. For example, an estimate of the cross-correlations between
the estimated parameters can be obtained from the correlation matrix. The
cross-correlation coefficients, between any two fitting parameters and
are defined as
where the k and j subscripts refer to elements of the correlation matrix and
the matrix.
The cross-correlation between fitting parameters, is particularly
useful to test the reliability of the determined parameters, If any of the
values of approach plus or minus one, then any variation in can be
almost totally compensated for by a variation of As a consequence, unique
values of and cannot be evaluated without additional information.
Unfortunately, the degree to which the can approach plus or minus one
without indicating a serious problem with the parameter estimation is not well
defined for nonlinear least-squares problems. The definition of the is
based on a number of limiting assumptions, such as the assumption that the
fitting equation was linear in the fitting parameters. Consequently, the use of
the cross-correlation coefficients is only approximate for nonlinear problems.

However, in a number of nonlinear problems it has been empirically observed
that an acceptable value for appears to be within approximately
Another use of the information matrix, (P´P), is to evaluate the variance–

covariance matrix whose elements are
One of the most common uses of the variance–covariance matrix is to use

its diagonal elements, as estimates of the variance of the
determined parameters, The definition of the variance–covariance matrix
is also based on a number of limiting assumptions, such as the assumption
that the fitting equation is linear in the fitting parameters. It has also been
assumed that the number of data points is large enough that the sample
variance, is a close approximation to the parent population variance,
Furthermore, the use of only the diagonal elements of the variance–covariance
matrix is equivalent to assuming that the parameters are orthogonal or
uncorrelated, that is, that a variation of one of the parameters, does not
effect the evaluation of the other parameters, for Consequently, the
use of the diagonal elements of the variance–covariance matrix as estimates of
the variance of determined parameters is only approximate for nonlinear
problems and for linear problems where the parameters are not orthogonal.
The square roots of these diagonal elements of the variance–covariance matrix
are sometimes called the asymptotic standard errors of the determined
parameters. A survey of methods for the evaluation of the joint nonlinear
confidence intervals of the determined parameters will be presented later in
this chapter.
4.4.2. Nelder–Mead Simplex Algorithm
The Nelder–Mead parameter estimation algorithm (6,13) provides a method

for minimizing, or maximizing, any NORM of the data and does not require
evaluation of derivatives or inversion of matrices. In the case of least-squares
minimization, this NORM is the sample variance, in Eq. (4.14). The
Nelder–Mead procedure involves a multidimensional search for the minimum
variance by performing a series of carefully selected one-dimensional searches.
This method always converges if a minimum exists. For the following
description of this method it is assumed that the variance of the residuals,
in Eq. (4.14), is being minimized.
The determination of n parameter values requires that n + 1 different sets
of parameter values be arbitrarily selected such that an n-dimensional simplex
with n + 1 vertices is defined. Each of these vertices is represented by a vector

of parameter values, which defines its coordinates. The superscript specifies
a particular vertex of the simplex. For example, for a problem in which
two parameters are being estimated, the simplex which is manipulated is a
triangle: three (n + 1) vertices in a two (n) dimensional space of parameters
being estimated.
Initial estimates for parameter values are required to begin the parameter
estimation procedure. A set of n + 1 simplex vertices is generated. These
vertices must be chosen such that they define an n-dimensional figure rather
than a line in n dimensions. One way to accomplish this is to choose the n + 1
vertices such that the arithmetic mean of the vertices corresponds to the initial
estimates of the parameters being determined and such that the vectors from
the initial estimates to the vertices are maximally orthogonalized. A judgment
must be made, however, when assigning appropriate ranges for each of the
parameters during generation of the initial simplex. An initial simplex
exhibiting either too closely spaced or too distantly spaced a distribution of
vertices may present the parameter estimation algorithm with a variance space
which causes insensitive or hypersensitive response behavior. The algorithm
may therefore incorrectly assume that convergence has been achieved, or it may
migrate to an undesired local minimum quite distant from the initial estimate
values. Some rationale, whether theoretical or empirical, must therefore be
invoiced to define the range of values which the initial simplex may span.
The first step in the estimation procedure involves calculation of the
sample variance at each of the n + 1 simplex vertices. The vertex with the
highest variance, is identified, and the centroid of the remaining vertices is
calculated. The centroid, is the average value of all vertices except the
vertex with the highest variance, A one-dimensional search is then con-
ducted along the line defined by the points and The immediate goal of
this search is to identify a new set of parameter values that yield a variance
lower than the maximum observed among the current simplex vertices. The
vertex of the current simplex exhibiting the highest variance, is then
replaced by this newly identified vector of parameter values, and then the
entire Nelder–Mead process is repeated.
Three operations are available for this one-dimensional search: reflection,
expansion, and contraction. Figure 4.3 presents a graphical depiction of these
three operations as applied to a two-parameter estimation problem.
The reflection operation generates a vector of parameter values, as
where is the reflection of the current simplex vertex with the highest
variance, relative to the centroid of the remaining vertices, and is the
reflection coefficient The reflected vector is thus on the line joining
and on the side opposite from If the variance, associated with

is smaller than the two highest variances and higher than the smallest vertex
variance, then is replaced with This generates a new simplex from which
the Nelder–Mead parameter estimation process is repeated.
Should the variance, of the reflected vector, be smaller than the
smallest vertex variance, then rv is expanded to as
where is the expansion coefficient If the variance, associated

with the expanded vector, is less than the variance associated with the
reflected vector, then is replaced with to generate a new
simplex and the process is begun again. If is greater than
the expansion operation has failed, is replaced with and then the
Nelder–Mead estimation procedure is repeated.
The reflection operation, however, may not yield a parameter vector with
an associated variance smaller than that of two of the current simplex vertices.
On such condition, a new is defined as either the old or as
if The contraction operation is then performed on the resultant
simplex as
where is the contracted vector, and is the contraction coefficient

If is replaced with thus generating an improved

simplex, and the Nelder–Mead parameter estimation process is repeated.
If the contraction also fails to reduce the value of a new simplex is
generated by replacing all of the simplex vertices, with for
i = 1, 2, 3, ..., n + 1, and then the parameter estimation procedure is repeated.
The vertex of the current simplex with the smallest associated variance is
represented by Upon encountering a failed contraction, the current simplex
is replaced by one in which all vertices have been contracted in the direction
of the vertex with the smallest currently identified variance.
The reflection, expansion, and contraction operations just described
involve one-dimensional searches over relative distances that are defined by
the magnitude of the respective operation coefficients. Optimal values for the
coefficients cannot be assigned based on any theoretical considerations. An
empirical approach was applied by Nelder and Mead (6) to a number of
analytically challenging response surfaces. The conclusion of their attempts is
that values of and produce optimum behavior from
the algorithm.
The criterion for convergence, that condition which signals the satis-
factory completion of the parameter estimation process, can conceivably be
defined in a number of ways. For example, when relative or absolute changes
in the values of the variable parameters among successive iterations become
smaller than some specified tolerance limit, convergence may be considered to
have been achieved. Such a criterion, however, implicitly requires knowledge
about the absolute or relative values of the variable parameters at or near
the minimum in variance space as well as about sensitivity of the variance
to changes in these parameters. A too stringent criterion may not signal
convergence until an unnecessarily large number of iterations is performed
or, in the extreme, may not permit termination of the parameter estimation
process at all, due to round-off and truncation errors. A too lax criterion,
on the other hand, may prematurely cause the algorithm to decide that
convergence has been achieved and perhaps report parameter values that are
a poor approximation to those characteristic of the true minimum in variance
space.
A criterion for convergence that may generally be applied, independent
of the values of the parameters being estimated or of the sensitivity of the
variance to changes in these parameter values, involves terminating the
estimation procedure when the relative change in the variance is less than
some specified value among successive iterations. Use of such a criterion for
convergence is a theoretically sound choice because, depending upon the extent
of curvature of the variance space at or near the minimum, the values of the
parameters in the fitting vector may be well defined (marked curvature) or
poorly defined (slight curvature). Terminating the estimation procedure
when the fractional change in the variance is 0.0001 or less generally ensures
identification of a variance minimum. This mechanism implicitly identifies the

minimum with sufficient accuracy to permit confidence in the derived parameter
values relative to their statistical uncertainty.
The Nelder–Mead simplex algorithm exhibits linear convergence behavior.
This simply means that the relative changes in parameter value for the
current iteration are approximately proportional to the changes in the
previous iteration. This is in contrast to the quadratic convergence behavior
displayed by the Gauss–Newton method in which the relative changes in
parameter values improve geometrically among successive iterations (see
Table 4.1). The more rapid convergence characteristic of the Gauss–Newton
algorithm arises from the use of derivatives of the function with
respect to the variable parameters, Calculation of derivatives and manipu-
lation of matrices, as in the Gauss–Newton procedure, are time-consuming
processes but permit convergence with fewer iterations than does the simplex
approach. A trade-off therefore occurs between the time required for
individual iterations and the number of iterations required before convergence
is achieved. The net computational time required to converge is almost always
less for the Gauss–Newton parameter estimation procedure as compared to
the Nelder–Mead simplex algorithm. However, derivatives of the function and
matrix inversions are not required by the Nelder–Mead procedure, thus
making this algorithm, in principle, somewhat easier to encode.
A major advantage of the simplex algorithm over most other parameter
estimation protocols is that any NORM may be employed in the estimation
process, not just the sample variance, This quality makes the Nelder–Mead
algorithm potentially useful in a variety of applications, even those in which
no explicit functional form is known. The following example describes an
application of the Nelder–Mead algorithm to maximize a NORM which is an
experimental response, instead of minimizing the least-squares NORM,
Identification of the conditions at which an enzyme exhibits its maximum
catalytic velocity as a function of the three variables pH, temperature, and salt
concentration may be accomplished by exhaustively assaying as a function
of each of these variables. This could be accomplished by searching pH,
temperature, and salt concentration at all possible combinations of ten
different values each, that is, as a search of a three-dimensional grid that is
dimensioned 10 × 10 × 10. This approach would require performing the assay a
thousand times to fully characterize the catalytic velocity with respect to each
variable, a process that would almost certainly involve expenditure of much
time and laboratory resources. An alternative approach would utilize the
Nelder–Mead simplex operations to guide the experimenter in deciding which
sets of conditions to examine. The experimenter can arbitrarily choose four
sets of values (n + 1 points in an n-dimensional space) for the three variables
pH, temperature, and salt concentration. The NORM of each of these sets
of values is evaluated by actually performing a laboratory experiment to
determine the enzyme’s catalytic velocity for each set of conditions. The
Nelder–Mead parameter estimation algorithm can then be applied to arrive
at the next set of conditions (pH, temperature, and salt concentration) for
an experimental determination of the catalytic velocity. The Nelder–Mead
process is continued with actual experimental measurements as the method of
evaluation of the NORM, catalytic velocity, to be maximized. Convergence is
achieved when the observed catalytic activity of the enzyme no longer changes
within the precision of the experimental observations. Such an approach will
permit identification of the desired optimal conditions with much less effort
than that involved in performing the assay at all of the possible one thousand
sets of conditions.
The Hessian matrix of second derivatives of the NORM with respect to
the parameters being estimated is analogous to the (P´P) matrix of the
Gauss–Newton procedure. This matrix can be utilized to evaluate the same
variance–covariance matrix and the cross-correlation coefficients of the
estimated parameters as with the Gauss–Newton procedure. A convenient
method for estimating the shape of the variance surface near the minimum
involves using a quadratic approximation.(14) The n + 1 vertices characteristic
of the final, converged simplex are used to create “half-way
points” defined as A quadratic surface is then estimated
from this combined set of (n + 1)(n + 2)/2 points. The original vertices of the
final, converged simplex are used in defining a set of oblique
axes with coordinates thus permitting the points to be represented as:
The quadratic approximation of this variance surface, near the minimum

is then given by
where the are elements of the vector A and the are the elements of
the matrix B, which is analogous to the (P´P) matrix of the Gauss–Newton
procedure. Equation (4.32) can be written in matrix notation, as
where the prime refers to the transpose of the matrix. The coefficients are
estimated as:
A set of parameters corresponding to the minimum variance may be estimated

by
In general, given a convergence criterion sufficiently stringent (less than

0.0001 relative change in variance, for example), any improvement in the
estimate of realized by Eq. (4.40) will not be significant relative to the
statistical uncertainty inherent in the derived parameter values. Additionally,
the information contained in the matrix B is based upon the assumption that
the variance near the minimum (as defined by the final simplex vertices and the
associated “half-way points”) may be described by a quadratic surface. Any
nonlinear deviations of the true variance surface from the assumed quadratic
behavior will introduce a systematic error into the statistical uncertainties
derived for the estimated parameter values.
Quadratic approximation demands careful consideration of possible
computer-associated round-off and truncation errors. Calculation of the coef-
ficients in the matrix B may be substantially biased by round-off errors when
the final simplex vertices (and “half-way points”) are too closely spaced.
It will therefore be necessary to enlarge the final simplex by expanding the
vertices away from the centroid until the values calculated for the and
are a factor of 1000 or more greater than the round-off error. Of course, a
trade-off occurs as a result of this expansion in that a more distantly spaced
simplex increases the probability that the variance space encompassed by the
simplex will deviate from a quadratic surface.
4.5. An Example of the Least-Squares Procedures—

Collisional Quenching
Collisional quenching of fluorescence requires contact between the

fluorophore and quencher during the lifetime of the excited state. In the
presence of quenching the decays of fluorescence intensity are expected to

become more complex than a single exponential due to transient effects which
occur immediately following excitation of the fluorophore. The detailed form
of the decay law is expected to depend upon the interaction radius, R, the sum
of the donor and acceptor diffusion coefficients. D, and the specific rate
constant, K, for quenching [see Eqs. (4.44)–(4.46) below]. Consequently, the
time-resolved decays reveal details of the interactions between donor and
acceptor in solution. Table 4.2 presents some actual experimental measure-
ments for the transient effects in collisional quenching of 1,2-benzanthracene
(BA) by 0.1 M carbon tetrabromide as measured by frequency-domain
fluorometry. (4) These data were taken from a concentration series which
was used to compare the classical square root of time decay law, Eq. (4.9),
with the more complete radiation boundary condition decay law. For more
details about the experimental protocol, refer to Joshi et al.(4) In the present
example we will analyze these data by the classical square root of time decay
law and the more complete radiation boundary condition formulation of the
decay law.(4)
The independent variable for this example is excitation frequency,
Each of the 21 data points has two different dependent variables,
amplitude modulation and phase shift, for a total of 42 experimental observa-
tions. The form of the fitting equations for these two dependent variables,
and is given by Eqs. (4.4)–(4.7) in terms of the intensity
decay law,
The fluorescence intensity decay, for the square root of time
approximation of the decay law is given by Eqs. (4.9) where
where is the fluorescence decay lifetime in the absence of quencher, R is the

interaction radius (the sum of the radii of the fluorophore and the quencher),
D is the sum of the diffusion coefficients, N' is (Avogadro’s
number/1000), and [Q] is the concentration of the quencher. Consequently,
for the square root of time intensity decay law, Eqs. (4.9), (4.42), and (4.43),
the vector of fitting parameters, has three elements: R, and D.
For the more complete radiation boundary model of collisional quenching,
the intensity decay, is given by
where the time-dependent quenching rate constant, k(t), is given by
where
erfc( ) is the error function complement, K is a specific rate constant for

quenching and has units of cm/s, is the fluorescence decay lifetime in the
absence of quencher, R is the interaction radius (the sum of the radii of the
fluorophore and the quencher), D is the sum of the diffusion coefficients, N'
is (Avogadro’s number/1000), and [Q] is the concentration of
the quencher. Consequently, for the radiation boundary condition intensity

decay law, Eqs. (4.44)–(4.46), the vector of fitting parameters, has four
elements: R, D, and K.
The reader should note that the actual fitting function, the combination
of Eqs. (4.44)–(4.46) for with Eqs. (4.4)–(4.7) for frequency-domain
fluorescence lifetime measurements, is a system of equations requiring evalua-
tion of a double integral. Neither of these integrals can be done explicitly, so
the integrals were performed numerically. The integral in Eq. (4.44) can be
written in the form
where x is defined by Eq. (4.46). A series of values of f(x) were evaluated once
and stored for 12 logarithmically spaced values of x. The error function
complement, erfc( ), was evaluated by the method of Hastings et al.(15) The
evaluation of Eqs. (4.44)–(4.47) utilized a cubic spline interpolation (Ref. 7,
p. 70) of the values f(x) and x for each iteration of the parameter estimation
process. The integrations in Eqs. (4.6) and (4.7) were performed by an 8-panel
Newton–Cotes adaptive quadrature routine, DQUANC (Ref. 7, p. 97).
The choice of units for the fitting parameters, is important to avoid
truncation and round-off errors during the calculations. Consequently, the
intrinsic fluorophore lifetime, was calculated in nanoseconds, the unit for
the interaction radius, R, was angstroms, the sum of the donor and acceptor
diffusion coefficients was expressed as the logarithm (base ten) of D in cm2/s,
and the specific rate constant, K, was expressed as cm/s.
For the purposes of this example we have actually only performed the
parameter estimation process on two parameters, the interaction radius, R,
and the log of the diffusion coefficient, log D. This was done to simplify the
problem so that two-dimensional graphs of the process could be presented.
The donor lifetime was assigned the value 38.8 ns and the quenching rate
constant was assumed to be 150 cm/s, as in the paper by Joshi et al.(4) Given
sufficient data, the least-squares procedures would, of course, be capable of an
evaluation of more than two parameters. The number of degrees of freedom
for this analysis is 40, that is, 21 data points times 2 dependent variables per
data point minus 2 parameters being estimated.
4.5.1. Example of the Gauss–Newton Procedure
In Table 4.3 the values of the interaction radius, R, and the logarithm of
the sum of the donor and acceptor diffusion coefficients, log D, are presented
for successive iterations of the Gauss–Newton procedure applied to the data in
Table 4.2 and the radiation boundary intensity decay law, Eqs. (4.44)–(4.46).
These results were presented in Table 2 of Joshi et al.(4) The rapid, quadratic
convergence properties of the Gauss–Newton procedure are clearly shown
in Table 4.3. The sample variance, decreased from 5605 to 98 in the first
iteration. By the fourth iteration the procedure has converged to where the
fractional changes in the parameters were 0.00005 for log D and 0.00018 for
R while the variance decreased by a fractional change of only 0.00018. The
changes for the fifth iteration were below the level of the single-precision
arithmetic of the computer. At each iteration the derivatives of the fitting
parameters were evaluated by a three-point Lagrange differentiation for-
mula, (12) so each iteration required five evaluations of the fitting function at
each data point.
Once the Gauss–Newton procedure has converged, the (P'P) matrix
can be used to evaluate several measures of the reliability of the parameter
estimation procedure. The cross-correlation coefficient between R and log D,
for this example, is –0.90280. This value is within an acceptable range,
indicating that the parameter estimation procedure has worked reasonably
well. The asymptotic variance of the estimate of the interaction radius, R, is
which corresponds to an asymptotic standard error of
The asymptotic variance of the estimate of the logarithm of the sum of the
donor and acceptor diffusion coefficients, log D, is which
corresponds to an asymptotic standard error of The asymptotic
covariance between R and log D was
The eigenvalues of the correlation matrix were 1.90280 and 0.09719545.
Neither eigenvalue is zero, indicating that the analysis procedure has
produced reasonable values. The largest eigenvalue is proportional to the
error in the asymptotic standard errors of the fitted parameters when cross-
correlation with other fitted parameters is neglected. In this case, the actual
standard errors of the determined parameters will be about 1.9 times the
asymptotic standard errors.
Table 4.4 presents a comparison of the values of R, D, and for the
square root of time and the radiation boundary intensity decay laws. (4) It is
clear that the values of the interaction radius and the total diffusion coefficient
as obtained from the two fitting functions, Eqs. (4.44)–(4.46) as compared to
Eqs. (4.9) and (4.42)–(4.43), are quite different. This is an example of a conse-
quence of the fourth assumption of least-squares parameter estimation. The
physical meaning of the determined parameters is based on an assumption
that the fitting equation, is correct. This assumption applies to all
parameter estimation procedures, not just least-squares. Consequently, the
determined mechanistic parameters should be reported only with reference to
the assumed mechanism.
It is also clear from this example that the variance of fit, is sub-
stantially larger for the square root of time intensity decay law as compared
to the radiation boundary condition intensity decay law. Thus, the radiation
boundary condition intensity decay law appears to be a better description of
the actual experimental data. If the two sample variances, in Table 4.4,
were only different by 10% instead of 480%, the statistical validity of this
statement would be questionable. An F test(16) can be applied to evaluate the
probability that the radiation boundary condition intensity decay law is a
better description of the actual experimental data than the square root of time
intensity decay law.
In the present example the F test is used as a measure of the probability
that the data analysis by the two independent intensity decay laws yields
residuals—the differences between the calculated values and the actual data
points—which are different. The F statistic is defined as the ratio of the two
variances:
where is the number of degrees of freedom of the variance in the

numerator, is the number of degrees of freedom of the variance in
the denominator, and PROB is the probability that and are dif-
ferent. For the present example, and
Most statistics textbooks contain tables of values
of the critical F statistic for different values of and PROB. (17)
Table 4.5 presents the value of the F statistic for for such
a series of probabilities. This table shows that the probability that the square
root of time and radiation boundary condition intensity decay laws yield
equivalent fits, for this particular data set, is vanishingly small.
An alternative to the use of tabulated values found in statistics books is
to generate a computer program which explicitly calculates the values in the
table. The reader is referred to Zelen and Severo(18) for the mathematical
formulas with particular reference to their equations 26.2.15, 26.2.16, and
26.2.23. A second alternative is to use the IMSL† subroutine MDFI, which finds
the inverse of the F probability distribution function.
4.5.2. Example of the Nelde–Mead Simplex Procedure
Table 4.6 and Figure 4.4. demonstrate the use of the Nelder–Mead simplex
procedure for the same radiation boundary condition example as used for the
Gauss–Newton procedure above. Points A, B, and C are the initial simplex
†
The IMSL is the International Mathematical and Statistical Library. It is the most complete
library of subroutines of its type and was written in standard FORTRAN. It is available from the
International Mathematical and Statistical Library (IMSL) Distribution Service, P.O. Box
4605, Houston, Texas 77210-4605.
vertices in this example, and the ellipse is a contour of constant variance,

about the maximum-likelihood values which are marked with a plus sign.
Point D is the reflection of vertex A, the current simplex vertex with the
highest associated variance, about the centroid of the remaining simplex
vertices, B and C. The variance associated with point D is lower than that
associated with any of the current simplex vertices, A, B, or C; consequently,
an expansion of point D to point E is attempted. Point E produces a further
improvement in the variance so that the expansion is successful and the
simplex for the next iteration is defined by the points BCE.
Vertex B now corresponds to the vertex with the highest associated

variance of the current simplex vertices. Point F is then calculated as the
reflection of point B about the centroid of the remaining vertices, C and E.
The variance associated with point F is less than that associated with two of
the current simplex vertices but greater than that of the third. The reflection
is therefore successful, and the simplex CEF becomes the simplex for the next
iteration.
Vertec C now has the highest associated variance, and point G is its
reflection about the centroid of E and F. The variance associated with point
G is greater than any of the variances associated with the current simplex
vertices. The reflection operation therefore fails, and a contraction is per-
formed. Point H is the contraction of vertex C toward the centroid of E
and F. The variance associated with point H is smaller than that associated
with two of the current simplex vertices and greater than that of the third.
Therefore, the contraction successfully leads to the generation of a new
current simplex, EFH.
This procedure is continued until the fractional change in variances
associated with the simplex vertices varies by less than 0.0001 (the convergence
criterion), thus signaling the end of the estimation process. Ultimately, the
final, converged simplex will closely surround the end point (marked by +).
The spacing between the vertices of this final, converged simplex will depend
upon the tolerance required to achieve convergence as specified by the
convergence criterion. To demonstrate the “average” improvement in the
estimated parameters per iteration, the centroids of the successive simplexes
deduced above are presented in the lower portion of Table 4.6. The variance
associated with these centroids is seen to be approaching steadily that
associated with the end point.
Once convergence has been reached the information matrix, B, can be
evaluated from Eqs. (4.36) and (4.37). This information matrix can then be
used to calculate the cross-correlation matrix, the variance–covariance matrix,
etc., as shown above for the Gauss–Newton procedure. A comparison of the
two methods indicates that both procedures reach the same values, within
reasonable precision, but the Nelder–Mead technique is slower.
4.6. Joint Confidence Intervals—Estimation and Propagation
The analysis of a set of experimental data by a procedure such as least-

squares involves not only the evaluation of the parameter values, which have
the maximum likelihood of being correct, but also the evaluation of a reasonable
estimate of the joint confidence intervals of those determined parameters. It is
the joint confidence intervals of the determined parameters which provide a
measure of the overall precision of the parameters and thus the reliability of
the estimated values. The joint confidence interval for a determined parameter
includes the effects of variations of all of the other parameters, within their
respective joint confidence intervals, while simultaneously maintaining a
statistically valid fit of the data. The maximum-likelihood parameter values
have little meaning without a reasonable estimate of their precision. Conse-
quently, the maximum-likelihood parameter values and the associated joint
confidence intervals should always be reported as a single entity; that is, never
report the parameter values without a reasonable estimate of their uncertainty.
4.6.1. Asymptotic Standard Errors
The simplest estimates of the precision of the determined parameters are

the square roots of the diagonal elements of the variance–covariance matrix,
Eq. (4.27). These provide an estimate of the asymptotic standard errors of the
determined parameters. However, as described earlier, the derivation of the
asymptotic standard errors is based on two limiting assumptions: (1) that
the fitting equation is linear, and (2) that the determined parameters are
orthogonal. The first of these assumptions means that the shape of the
variance space, that is, the values of the variance in the neighborhood of the
maximum-likelihood values, can be predicted from the minimum variance and
the (P'P) matrix from the Gauss-Newton procedure or the B matrix from the
Nelder–Mead procedure. When parameters are orthogonal, the determination
of a particular parameter, is independent of the determination of all of the
other parameters, and all of the off-diagonal elements of the (P'P) matrix
are zero; that is, the parameters are not correlated. In general, these two
assumptions are unlikely to be satisfied. Consequently, a more realistic
estimate of the confidence intervals of the determined parameters is needed.
4.6.2. Linear Joint Confidence Intervals
A well-known linear approximation to the joint confidence interval is

provided by the solutions, . of
where n is the number of parameters, s2 is the estimated variance of the

minimum, F-statistic is based on the desired confidence probability and the
number of degrees of freedom, and P, P', and are as previously defined.(19-22)
The solutions, of Eq. (4.49) predict a multidimensional elliptically shaped
contour in a parameter space which corresponds to a constant confidence
probability. The elliptically shaped region in Figure 4.4 is a two-dimensional
example of a joint confidence contour. It should be noted that the ellipse in

Figure 4.4 was actually evaluated by exhaustively searching the parameter
space for a 67% confidence level, rather than by evaluating the solutions of
Eq. (4.49), and thus represents the exact confidence contour. In this case,
however, the ellipse in Figure 4.4 is a close approximation to the solution of
Eq. (4.49). Any pair of values, log D and R, which are inside this contour are
acceptable within the specified probability level, and any pair of parameters
outside the contour are excluded at that probability level. Consequently, the
maximum range over which either of the parameters, log D or R, can deviate
from the optimal values and still remain within the joint confidence region
is used as an approximation of the confidence region for that parameter.
This approximation to the joint confidence interval correctly considers the
nonorthogonal nature of the determined parameters, but fails to consider the
nonlinear nature of the fitting equation and consequently the nonlinearity of
the variance space.
4.6.3. Support Plane Confidence Intervals
It is possible to determine a confidence interval for each of the deter-

mined parameters by the support plane procedure. For this procedure a series
of values for a particular determined parameter are assumed. For each of
these assumed values the optimal values of all of the other determined
parameters are redetermined by nonlinear least squares, and the variance at
each of the series of assumed values is evaluated. This procedure thus creates
a table of values for the optimal variance as a function of assumed values for
one of the determined parameters. This series of variance values can now be
interpolated to determine where a statistically significant increase in the
variance occurs. The points at which these statistically significant increases
occur mark the ends of the confidence interval for that determined parameter.
The process is repeated for each of the determined parameters.
A significant increase in the variance is defined by partitioning the
variance into two terms:
where S2minimum is the sample variance at the maximum-likelihood parameter

values, s2 is the sample variance at any other set of parameter values, and
S2parameters is that por tion of the total variance which can be attributed to a
variation in the parameter values. The number of degrees of freedom for
S2minimum is the number of observations minus the number of constraints,
that is, the number of data points times the number of dependent variables
per data point minus the number of determined parameters, N – NDP. The
number of degrees of freedom for S2parameters is just the number of determined

parameters, NDP. The critical ratio of these variances, F-statistic, for any
probability level, PROB, is determined by
where F(NDP, N – NDP, 1 – PROB) is the F-statistic for NDP degrees of

freedom, N – NDP degrees of freedom, and a probability of 1– PROB. This
translates into a fractional increase in the overall variance due to a variation
of the parameters as
For a one-standard deviation confidence interval the value of PROB is

approximately 67%. For a two-standard deviation confidence interval the
value of PROB is approximately 95 %.
Figure 4.5 presents a support plane analysis of the radiation boundary
condition collisional quenching example presented in Figure 4.4 and
Tables 4.2–4.4 and 4.6. This figure is a rotation of the ellipse in Figures 4.4
and 4.6 into a third dimension, showing a cross-sectional cut. A series of
values of the interaction radius, R, were chosen to span the maximum-
likelihood value. For each of these values the maximum-likelihood value of
the logarithm of the sum of the donor and acceptor diffusion coefficients,
log D, and the corresponding variances were determined by nonlinear least
squares. Figure 4.5 presents a graph of the variance at the maximum-likeli-
hood value of log D for each of the assumed values of R. For this example
two parameters were determined, there were 21 data points, each with two
dependent variables, and F(2, 40, 1.0–0.67) 1.14. Consequently, the critical
value of the fractional increase in the overall variance, from Eq. (4.52), is
approximately 1.057. The horizontal line in Figure 4.5 corresponds to a frac-

tional change of 1.057 in the overall variance of fit introduced by a variation
of the interaction radius. The points of intersection of this horizontal line with
the variance curve correspond to the end points of a 67 % confidence interval.
The vertical lines represent the projection of these points of intersection onto
the interaction radius axis; that is, 8.386 interaction radius 8.562.
In recapitulation, the support plane procedure correctly treats nonlinear
variance spaces and nonorthogonal determined parameters. Its major limita-
tion is the large amount of computer time required to evaluate the confidence
limits. For example, consider a hypothetical complex problem where six
parameters are simultaneously being estimated. If ten values of the variance as
a function of each of the determined parameters are required to determine the
confidence interval of each determined parameter, then the group of six
parameters will require 60 variance values. Remember that each of these
variance values is determined by a five-parameter least-squares parameter
estimation procedure. Therefore, for this hypothetical example the amount of
computer time required to evaluate the confidence intervals by the support
plane procedure is approximately 60 times the amount of computer time
required to determine the actual maximum-likelihood values of the parameters.
Clearly, a method which requires less total computer time is desirable for
complex problems.
4.6.4. Approximate Nonlinear Support Plane Joint Confidence Intervals
The only disadvantage of evaluating the confidence intervals by the

support plane method is the large amount of computer time required for
a complex multiparameter analysis. The disadvantage of the linear joint
confidence interval method is that it does not consider the nonlinear nature
of the fitting equations and consequently can, in some cases, provide incorrect
estimates of the confidence intervals of the derived parameters. We have
developed an approximate method for the evaluation of nonlinear joint con-
fidence intervals which is, in effect, a combination of the support plane
method and the linear joint confidence interval method.(5,20–22)
The support plane method is time-consuming because each of the fitted
parameters must be searched independently for a critical variance ratio
(Eq. 4.52), and each point on this search requires that the least-squares fitting
procedure be repeated in order to account for the possible variation of all of
the other fitting parameters. If the variation required to maintain the lowest
possible variance in the other fitting parameters can be predicted a priori, then
only a single search would yield a reasonable approximation of the joint
confidence intervals for all of the parameters in question. Furthermore, each
of the variance estimates required for this search can be directly evaluated by
Eq. (4.14), and the entire least-squares procedure need not be repeated. Thus,
if the joint variation in the fitting parameters for a minimum variance can be
predicted a priori, then the support plane method for the evaluation of the
confidence intervals is practical for multiparameter problems.
The advantage of the linear joint confidence interval method is that, for
a linear fitting equation, it provides a method of predicting the shape of
the variance space for any variation of the parameters. The major axis of
the multidimensional elliptically shaped solution of Eq. (4.49) is a good
approximation, even for most nonlinear problems, of how the variation
of a single fitting parameter will induce a variation in the remaining fitting
parameters and still maintain the lowest possible variance. Consequently, the
solutions of Eq. (4.49) can be used to vary the parameters jointly in the
support plane method for multiparameter problems. The reader is cautioned
that this approach is only an approximation to the complete support plane
method, but our experience has been that it is almost always a very good
approximation.
The solutions, of Eq. (4.49) define a new set of fitting parameters.
These new fitting parameters, NEW i , are formed as
where the summation is over each of the n fitting parameters, and the SFij are
a set of scaling factors. These scaling factors are chosen such that the new
parameters, NEW i , are all orthogonal. Since these new parameters are all
orthogonal to each other, the variation of one of these new parameters,
NEW i , will not affect the values of the other new parameters, NEW j , for
which correspond to a minimum variance.
The geometric interpretation of this new set of parameters is relatively
straightforward. Sets of parameter values which can be considered as “correct”
answers to within some statistical probability lie within the multidimensional
elliptical region (Figures 4.4 and 4.6) while sets of parameter values out-
side this elliptical region are considered to be “incorrect.” The axes of this
multidimensional ellipse do not, in general, coincide with the axes of our
coordinate system. The process of choosing a new coordinate system is simply
a rotation of the coordinate system such that the new coordinate system,
NEW i , coincides with the major and minor axes of the multidimensional
ellipse (as in Figures 4.4 and 4.6).
The support plane method can be applied to the variation of each of
these new parameters, NEW i , in order to find the value of each of the new
parameters which corresponds to the critical variance ratio (Eq. 4.52). This
is equivalent to searching each of the major and minor axes of the multi-
dimensional elliptically shaped confidence region (see Figures 4.4 and 4.6) for
the critical variance ratio. The values of the original fitting parameters, at
the ends of the axes of the elliptically shaped region can be evaluated from the
scaling factors, SFij, and the new parameters, NEW i , which correspond to
the critical variance ratio defined in Eq. (4.52). The maximum variations of
the original fitting parameters, are then taken as an approximation to the
nonlinear joint confidence intervals.
Figure 4.6 graphically depicts an example of this search procedure. It
corresponds to the analysis of the confidence intervals for the example which
was previously used to demonstrate the Gauss-Newton and the Nelder–Mead
procedures (see Figure 4.4 and Tables 4.2–4.6). The example involved the
evaluation of log D and R by the radiation boundary model based on the
data presented in Table 4.2. The elliptical region in Figure 4.6 is the same as
the region in Figure 4.4 and corresponds to a one-standard deviation, 67%
confidence region. The diamonds are the results of the search of the new
parameters NEW i which corresponded to the major axis of the solution of
Eq. (4.49). The triangles are the results of the search of the new parameters
NEW i which corresponded to the minor axis of the solution of Eq. (6.49). The
plus signs correspond to the values found by searching the original fitting
parameters, independently of each other, that is, without repeating the
fitting procedure at each of the values of currently being evaluated.
It is recommended that confidence regions be defined by the evaluation
of all of the points shown in Figure 4.6. This will require searches for the
critical variance ratio in 4n directions, where n is the number of parameters
being estimated, that is, each in both directions and each NEW i in both
directions. The reader is reminded that these 4n searches involve only the
evaluation of the variance; that is, they do not require additional least-squares
parameter estimation at every point in the search. Consequently, even though
this represents twice as many searches as the support plane method, each of
the searches is much faster. It is important to perform each of the searches in
both directions, because for nonlinear functions the joint confidence interval
is usually asymmetrical.(5, 20–22) The net result of performing all 4n searches is
that the entire shape of the joint confidence region is characterized, even when
it is asymmetrical.
The evaluation of the scaling factors, SFij, is relatively easy. The eigen-
vectors of the correlation matrix, Eq. (4.26), define the directions of the axes
of the multidimensional elliptical region in the cross-correlation space. By
multiplying the elements of these eigenvectors times the square roots of the
respective diagonal elements of the variance–covariance matrix, the eigen-
vectors become direction vectors in the original parameter space. It is these
direction vectors starting with the maximum-likelihood values of the fitting
parameters, which are to be searched for a critical variance ratio. The
eigenvalues provide the relative lengths of the axes. Thus, in order to search
the major axis for a critical variance ratio Eq. (4.52), the following steps need
to be performed. First, find the eigenvector of the correlation matrix that
corresponds to the largest eigenvalue of the correlation matrix. Second,
convert the eigenvector into the parameter space by multiplying its elements
by the particular eigenvalue and by the square roots of the respective diagonal
elements of the variance–covariance matrix. Third, search along this eigen-
vector for the set of parameter values which yield the critical variance ratio
calculated from Eq. (4.52). It should be noted that the shape of the variance
space for this search is proportional to the square of the distance along the
eigenvector. Be sure to search the eigenvector in both directions since the
variance space will, in general, not be symmetrical. Furthermore, it is
important to search all of the eigenvectors, that is, the eigenvectors which
correspond to all of the eigenvalues of the correlation matrix (see Section
4.6.6). The reader is referred to the EISPACK software package(8,9) for a group
of FORTRAN subroutines, such as TQL2 and TRED2, which can be used to
evaluate the eigenvalues and corresponding eigenvectors of the correlation
matrix.
In recapitulation, this approximate method for the evaluation of the
confidence intervals of determined parameters has the advantages of being
relatively fast, of considering the nonlinear nature of the fitting function,
and of considering the cross-correlation between the fitting parameters.
Although it is only an approximation of the support plane method, it is highly
recommended.
4.6.5. A Monte Carlo Method for the Evaluation of Confidence Intervals
The last method for the evaluation of confidence intervals which we will
present is a Monte Carlo method. This method has the advantage that it
directly generates the entire probability distribution for each of the determined
parameters without making an assumption about the shape of the probability
distribution. However, this method requires a knowledge of the shape of the
distribution of the experimental uncertainties associated with each of the data
points. It should also be noted that this method requires a large amount of
computer time and, as a consequence, probably should only be used in cases
where the entire probability distribution of the fitted parameters is required.
The basic method is very simple. The first step is to analyze the data by
any method, such as least-squares, which will yield a set of fitted parameters,
which have the maximum likelihood of being correct. Given this set of
answers, and the original distribution of independent variables, Xi, a set of
synthetic data is generated. This set of data simply consists of the evaluation
of the fitting function at and each of the Xi, The next step is to take this
perfect simulated data set and add to it realistic simulated experimental
uncertainties such that we have a data set which is based on a known set
of parameters and contains realistic experimental uncertainties. The con-
sideration of uncertainties requires the generation of pseudo-random noise
to be superimposed upon the simulated dependent variable values, Yi. The
particular distribution of this pseudo-random noise should be consistent with
the experimental uncertainty distribution of the actual experimental data. This
distribution of experimental uncertainties is required to follow a Gaussian
distribution by the second basic assumption of the least-squares method.
For a discussion of how to generate Gaussian distributed pseudo-random
numbers, see Ref. 7, p. 240. This last step is repeated a few hundred times such
that a few hundred simulated data sets are generated, each with a different set
of random uncertainties included. The least-squares analysis is then performed
on each of the few hundred simulated data sets, and frequency histograms of
each of the parameters, are generated from the results of the few hundred
least-squares parameter estimations. These histograms will represent a
reasonable approximation of the probability distributions for the determined
parameters based on an analysis of the original experimental data.
In effect this process has asked the question, “How does a particular dis-
tribution of experimental uncertainties affect the analysis of a particular data
set?” The results are dependent on the assumed magnitude and distribution of
experimental uncertainties. The results are also dependent on the particular
distribution of independent variables, Xi. As the number of simulated data
sets is increased, the precision of the resulting probability histograms will
increase. The exact number of simulated data sets which are required is
impossible to predict a priori, but a reasonable number seems to be a few
hundred. This, of course, means that the computer time required to perform
this process will be a few hundred times what was required for the original
least-squares analysis.
4.6.6. Propagation of Confidence Intervals

Quite often an investigator will wish to use a set of determined
parameters, and their associated confidence intervals to calculate some
other derived quantity. For example, for the square root of time description
of collisional quenching the investigator may wish to calculate the value of
from Eq. ( 4.42 ) or the value of b from Eq. (4.43). For the radiation boundary
example the investigator might want the value of the quenching rate at time
zero, k(0), from Eq. (4.45). It is relatively easy to calculate the maximum-
likelihood values of these quantities from the maximum-likelihood values of
the fitting parameters, However, the method for the propagation of the
confidence intervals is not obvious since the individual fitting parameters,
are expected to be correlated with each other and their respective confidence
intervals are expected to be asymmetrical.
The confidence intervals of the fitting parameters can be evaluated
relatively simply by the method outlined in Section 4.6.4.(22) The main
objective of this method is to characterize the shape of the one-standard
deviation joint confidence intervals by evaluating 4n points on the elliptically
shaped confidence region. A similar confidence region exists for any derived
quantity. All that is required to determine confidence intervals for any derived
parameter is to map each of the 4n points which characterize the original
confidence ellipse into a parameter space for each of the derived quantities.
This is done by evaluating the derived quantity at each of the 4n points which
describe the original confidence contour. The extreme spread of the resulting
4n values of each of the derived quantities is simply the one-standard
deviation confidence interval for that derived parameter.
Evaluating confidence intervals of derived parameters from the proba-
bility distributions obtained by the Monte Carlo method is also simple. The
derived parameters are evaluated from each set of the maximum-likelihood
fitted values, obtained from each of the few hundred sets of synthetic data.
A complete probability distribution of the resulting derived parameters can
then be constructed.
4.7. Analysis of Residuals
Estimation of parameters characteristic of some mathematical model by

a least-squares procedure produces those parameter values, which have the
greatest probability (i.e., maximum likelihood) of correctly accounting for the
experimental data being analyzed when certain assumptions are made about
the distribution of random experimental uncertainties. Before any quantitative
least-squares analysis of experimental observations is possible, however,
some reasonable fitting function, G must be generated. The process
of defining such a mathematical model involves theoretical considerations
and, often, previous experimental conclusions when the understanding of the
phenomenon being examined is evolving to a level of greater complexity.
Recent advances responsible for expanding the base of biophysical knowledge

as well as the ongoing development of more sophisticated instrumentation
have made necessary this evolution of mathematical models to account for the
more accurate and precise experimental observations now possible. Fewer
analytical approximations are necessary, and, in fact, approximations are
often no longer tolerable as progress in experimental and analytical methods
proceeds at an accelerating pace. Higher quality biophysical data obtained
at greater levels of determination often point out deficiencies in current
analytical models as evidenced by their inability to satisfactorily describe the
available data. The current section of this chapter deals with the methods
available for determining the appropriateness of an analytical model. This
may be accomplished most easily by examining the residuals.
Residuals are the differences between the observed experimental data and
the fitting function evaluated at the maximum-likelihood values of the
parameters, The residuals may be thought of as the observed experimental
uncertainties when the model is correct. As stated earlier (see Section 4.3),
certain assumptions are implicit and must necessarily hold if a least-squares
estimation procedure is to be a valid method for obtaining estimated model
parameter values. A way of demonstrating the consistency of a model in terms
of accounting for observed experimental behavior is to examine the residuals
in order to confirm or deny these assumptions. After examining the residuals,
a decision should be possible as to whether the assumptions are consistent
with the analysis. If examination of the residuals indicates that the assumptions
appear to be satisfied, they provide no justification for rejecting the current
model, G as an accurate description of the observed experimental
behavior. This is not the same as saying that examination of the residuals
justifies accepting the current model as necessarily being correct. The best one
can say in this case is that the model is consistent with observation. If, on
the other hand, the residuals are clearly inconsistent with the experimental
uncertainties of the experimental data, then the current model may correctly
be rejected.
Current analytical models and numerical methods are rapidly moving in
a direction of increasing complexity. As a result, very sophisticated means are
often employed to ultimately obtain estimated parameter values and residuals.
An investigator who is involved in analyzing complex biophysical phenomena
is obviously in a highly quantitative environment. However, when considering
the properties of residuals the investigator is required to operate in a largely
qualitative or, at best, semiquantitative domain. This arises due to the nature
of residuals and the means by which information may be extracted from them.
Plots of residuals versus a number of relevant parameters can be inspected
visually to recognize significant behavior. Cumulative frequency plots, plots
testing for serial correlation, and plots making possible detection of outliers
(bad points) all require such visualization as a basis for arriving at conclusions.
Some quantitative tests also exist permitting examination of the number of

runs (the number of consecutive positive and negative residuals) or detection
of influential observations (those which strongly influence the value of one
or more model-dependent parameter values). There are also the familiar
chi-square tests (for testing frequency distributions and for determining levels
of significance), the Durbin–Watson test (a test for a particular type of serial
correlation), and the Kolmogorov–Smirnov test (a goodness-of-fit test that is
an alternative to the chi-square test for frequency distributions). These are
just a few of the means available for analyzing residuals, some qualitative
and others quantitative. A number of these methods should be applied in
analyzing residuals in order to thoroughly examine the displayed behavior.
4.7.1. Plots
Visualization of residuals plotted as scatter diagrams against various

experimental parameters addresses two important points: whether one or
more of the assumptions of least-squares parameter estimation are violated
and whether correlations or trends are suggested in the behavior of the
experimental observations which have not been accounted for by the fitting
function used to analyze the data. Plots of the residuals, the observed minus
the calculated values of the dependent variable, are commonly generated
versus (a) Ycalc, the calculated values of the dependent variable, (b)X i , the
values of the independent variable(s), some potentially signifi-

cant functional relationship(s) of one or more of the independent variable(s),
and (d)X', one or more new independent variable(s) that potentially may be
correlated with the observed phenomenon but were not explicitly considered
in the current analytical model (Ref. 23, pp. 316–319).
Visual inspection of scatter diagrams of residuals versus and
[cases (a) and (b) above] permits immediate recognition of obviously non-
Gaussian residual distributions. The pseudo-random distributions presented
in Table 4.7 represent examples of approximately Gaussian and clearly non-
Gaussian distributed residuals as a function of an arbitrary abscissa (X axis).
Figure 4.7 is a graphical representation of the scatter diagrams for these same
two distributions. The point distribution in the scatter diagram of the upper
panel of Figure 4.7 shows no obvious non-Gaussian behavior, whereas the
lower panel of Figure 4.7 clearly exhibits an upward trend in the residual
values with increasing abscissa (X axis). If the variability in the residuals is
not constant, the experimenter is readily made aware of the presence of some
potential systematic behavior in the observed data. Some inadequacy of the
analytical model is implied if the variance of the residuals (proportional to the
width of the scatter band) remains approximately constant but the mean
residuals appear to show some trend with or This phenomenon is
shown in the lower panel of Figure 4.7. Differences in the variance of residuals
as a function of or on the other hand, imply that some potential
systematic behavior is arising from the measuring process itself rather than
from an inappropriate model used in analysis. Of course, combinations of
both of these effects may be present. This very simple, visual tool may there-
fore yield substantial insight into difficulties with the experimental procedure
and/or the fitting function.
Plots of the residuals versus [case (c) above] offer a test of the
hypothesis that the functional relationship(s), of one or more independent
variables, represents implied, or necessary, functional behavior that
should have been, but was not, included in the analytical model currently
employed in estimating the observed behavior. For example, suppose our
model is given by
where the two independent variables, and are assumed to be related

linearly to the observed data, by the variable parameters and The
residuals may be plotted against the product to test for any apparent
correlation of the data with this particular functional relationship (the
product) of the two independent variables. If, for example, a linear correlation
of the residuals with the product is apparent (as illustrated by the lower
panel of Figure 4.7), an additional term should be included in the current
fitting function, thus generating the modified function:
Although this example is a particularly elementary one, it serves to

demonstrate the potential usefulness of checking for correlation of the
residuals with some functional relationship(s) of the independent variable(s).
The mathematical complexity of analytical models will vary depending upon
the phenomenon, or combinations of phenomena, occurring and implicit in the
experimentally observed parameters with respect to the independent variable(s).
A thorough appreciation of the experimental system and phenomena being
studied may, however, suggest relationships worth considering in an exami-
nation of residuals based on theoretical and/or intuitive reasons and may

conceivably involve rather complicated mathematical formulations. The
reader is cautioned that including relationships which are based solely on
observed correlations but are without theoretical and/or intuitive reasons will
likely produce erroneous and/or irrelevant results with little physical meaning.
Therefore, the use of these plots represents a rather convenient, qualitative
way to test for some additional relevant effects not accounted for by a current
fitting function.
A concept closely related to the one just presented involves examining a
scatter diagram of the residuals plotted against some new independent
variable(s), X' [case (d) above]. The parameter X' is not explicitly considered
in the current analytical model but may play a role in producing the behavior
observed in the experiment. Any correlation apparent in a plot of residuals
versus X' suggests that X' should not be excluded from consideration.
A brief comment should be made about quantitative interpretations that
may be implied by the above scatter diagrams. Quantitative information
extracted from plots of residuals versus or X', for example, must be
viewed with caution. Such an examination will almost certainly accurately
suggest a valid qualitative relationship overlooked by the current analytical
model. However, the best quantitative estimate of the influence of any
particular independent variable should be considered simultaneously with all
other independent variables in one mathematical formulation of the analytical
model. Returning to the example used previously, suppose Eq. (4.54) is used
to analyze a set of experimental data and the residuals suggest the need for
an additional term such as that introduced in the modified function of
Eq. (4.55). If the third term, is completely uncorrelated with either
of the first two terms, and then estimating by fitting
to a plot of the residuals versus will yield an accurate estimate
of while and remain unchanged. If, however, some correlation exists
among and and/or (which in practice will almost always be the case
to at least some extent), this process will not yield the best least-squares
estimate of all three parameters. Instead, the modified fitting function must be
used to reanalyze the original data set while simultaneously estimating all
three variable parameters, and This process will permit effects of
correlation among the three fitting parameters to be accounted for during
the least-squares estimation procedure. Only this latter approach will permit
valid estimation of the three maximum-likelihood values for and
consistent with the expanded fitting function.
4.7.2. Distributions
Another graphical method useful for examining residuals is the cumulative

frequency plot.(24) This technique permits investigation of the probability
distribution of the residuals. The residuals must be ordered and numbered

such that:
with
The quantity is an estimate of the probability of encountering a residual

with an absolute value less than or equal to Plotting versus
approximates the cumulative distribution function of the residuals. Such a
plot, when generated on normal probability paper, will produce a straight line
if the residuals follow a Gaussian distribution. Converting the cumulative

probability, to its associated Z value (standard normal deviate) makes
generating the cumulative frequency plot more convenient in that the ordinate
(Y axis) is linear in Z-value space (for Gaussian distributions). Z values and
their associated probabilities may be found tabulated in most statistics books.
Table 4.8 and Figure 4.8 present the cumulative frequencies, associated Z
values, and ordered residuals for the residual distributions of Table 4.7 and
Figure 4.7. The left panel of Figure 4.8 (corresponding to the approximately
Gaussian distributed residuals of Table 4.7) shows all of the points residing
very near the theoretical cumulative distribution function line (for Gaussian
distributed residuals exhibiting a standard deviation of 0.92). This behavior
is expected if the residuals are truly Gaussian distributed. The right panel
of Figure 4.8 presents the cumulative frequency distribution for the non-
Gaussian distributed residuals of Table 4.7. The points for this case are not as
closely superimposable onto the theoretical line (again for a standard devia-
tion of 0.92) as was observed in the left panel. However, there would be little
justification for claiming that the distribution represented in the right panel
of Figure 4.8 is not Gaussian distributed on the basis of this test alone. The
presence of a few points distant from what is otherwise a good representation
of a straight line by the majority of points suggests the occurrence of outliers,
or bad points, a topic to be discussed later. Inability to generate a straight line
in a cumulative frequency plot indicates that the residuals are not Gaussian
distributed and thus represents a violation of one of the assumptions of the

least-squares method.
Another more quantitative method of assessing whether or not residuals
follow a Gaussian distribution involves evaluating the statistical probability
that the observed distribution is different from a Gaussian distribution
(Ref. 23, pp. 391–393, and Ref. 25, pp. 340–345). The range of residual values
is divided into mutually exclusive, continuous intervals, and the frequency of
occurrence of residuals possessing values within each of these intervals is
noted. The variance of the expected population of residuals is approximated
by the computed variance of the residuals, and the mean of the residuals is
assumed to be zero. This expected behavior of the distribution of residuals
then permits calculation of the expected frequencies in each of the above-
mentioned intervals if the distribution of residuals is truly Gaussian.
With this information in hand, the expected frequency of occurrence for
each interval may now be calculated. This involves determining the relative
frequency for each interval and multiplying it by the total number of residuals.
This is most easily achieved by working with standard normal deviates, Z,
and finding appropriate values from published tables of areas under standard
normal curves. Here, Z is defined as
where is the mean value (in this case, zero), s is the standard deviation of
the residuals (the square root of the sample variance), and X is one end of the
interval being considered. The absolute value is used to eliminate the
occurrence of negative Z values which would arise when For example,
suppose that the relative frequency of occurrence is desired within the interval
that is, for residuals with values greater than and less than
Values for and are calculated as per Eq. (4.59) by substituting or
for X. The relative frequency of occurrence over the interval will then
be defined by the expression where freq corresponds
to the frequency found from a table of areas under standard normal curves at
The absolute value is again used to eliminate the occurrence of
negative values. This process is carried out for all intervals, and the relative
expected frequencies are multiplied by the total number of residuals to yield
the absolute expected frequencies.
The next step involves quantitatively characterizing the discrepancies
between observed and expected frequencies by calculating a chi-square statistic
as
where and are the observed and expected frequencies in interval i,

respectively. If the residuals are actually Gaussian distributed, then this statistic
will be distributed approximately as given in chi-square tables (located in most
statistics texbooks) with (n – c) degrees of freedom, where n is the total number
of intervals and c is the number of constraints on the system. One constraint
arises from restricting and additional constraints occur for each
parameter estimated. In this case, only one additional constraint occurs from
estimation of the variance of the residuals, thus defining the value of s in
Eq. (4.59). The value of this chi-square statistic is then compared with
tabulated values of degrees of freedom] to quantify the
probability that the residuals do not follow a Gaussian distribution at the
level of confidence specified in the table used.
Table 4.9 shows the procedure and calculations necessary to apply this
test to the residual distributions of Table 4.7 and Figure 4.7. The range of
residual values was divided into nine equally spaced intervals, each of which
was (s/2) units wide. Here, s corresponds to the calculated standard deviation
of the residuals, which was approximately 0.92 for each of the distributions of
Table 4.7. The results of this analysis indicate that the approximately
Gaussian distribution has a probability of between 1% and 2.5% of
being non-Gaussian whereas the non-Gaussian distribution has a 75-90%
probability of being non-Gaussian. These probabilities are derived from the
calculated values with seven degrees of freedom.
A point must be made about small expected frequencies, that is, those
less than one. This approximation of is not strictly valid when the expected
frequencies are small. Generally, adjacent intervals should be combined to
achieve a desired minimum frequency of not less than one.
4.7.3. Trends
Another quality of residuals to be concerned with is the presence of

trends in the residuals with respect to dependent or independent variables.
Theoretically, it is desirable to have no correlation of residuals with respect to
any of these variables. The presence of trends suggests that some systematic
behavior is influencing the experimental observations and that this systematic
behavior is not being accounted for either due to (1) inappropriate experi-
mental design or (2) deficiencies in the fitting function.
An easy and convenient test for randomness of residuals with respect to
any variable is accomplished by counting the number of runs. (24) A run here
means a consecutive sequence of residuals of equal sign. If is the total
number of positive residuals and is the total number of negative residuals,
the number of runs expected, if the sequence of residuals is random is given
by
The variance of the number of runs, is given by
A parameter, Z, may then be defined as
where is the actual number of runs observed. The value of Z will be dis-
tributed approximately as a standard normal deviate if both and are
greater than 10. The value of 0.5 is a continuity correction which partially
compensates for the approximation of a discrete distribution by a continuous
distribution. If too many runs, instead of too few, are being tested for, a value
of –0.5 is used. This approach therefore permits one to estimate the statistical
probability that the number of runs actually encountered is different from that
expected if the residuals had occurred randomly with respect to the variable
under consideration. In other words, the greater the value of the greater
the probability that some correlation exists in the residuals relative to the
variable being considered.
Table 4.10 demonstrates an application of the runs test to the residual
distributions presented in Table 4.7 and Figure 4.7. The derived Z values
(between 0.40 and 1.24) are not sufficiently large to necessarily conclude that
either of these two distributions is not Gaussian distributed, based on this test
alone. On the other hand, values of Z greater than approximately 2.5 will
correspond to probabilities of less than about 1 % that the distributions are
actually random. It is important to note that a Z value greater than 2.5
implies that one may justifiably reject the hypothesis that the residuals are
Gaussian distributed. However, if a Z value less than or equal to 2.5 is
obtained with the runs test, one cannot necessarily conclude Gaussian
distribution of residuals without further confirmation.
Results of an analysis of the number of runs may indicate that too few
runs are present (implying possible positive serial correlation) or that too
many runs are present (implying possible negative serial correlation). The
term “serial correlation” implies a repeated pattern among residuals. Serial
correlation is most frequently applied to time series experiments. Variables
other than time, however, may conceivably offer practical significance with
regard to the concept of serial correlation under appropriate conditions.
Correlation among residuals may be determined 1,2,..., n units apart,
thus characterizing the lag1, lag2,..., lagn serial correlations, respectively.(26)
An examination of the lagn serial correlation of residuals is accomplished by
generating a plot of the value of each residual versus the value of the residual
occurring n units before the one currently being considered. Of course, the
first n residuals therefore cannot be plotted. Positive lagn serial correlation will
228 Martin Straume et al..
be apparent in a lagn serial plot as a band of points generally exhibiting a

trend from the lower left (negative, negative) quadrant to the upper right
(positive, positive) quadrant. Negative lagn serial correlation, on the other
hand, will be visualized as a band of points distributed from the upper left
(negative, positive) quadrant to the lower right (positive, negative) quadrant.
Residuals displaying no lagn serial correlation will produce a lagn serial plot
with no apparent trend. The residual distributions of Table 4.7 and Figure 4.7
are presented as lag 1 , lag2, lag3, and lag4 serial plots in Figure 4.9. Only the
lag1 serial plot for the non-Gaussian distributed residuals (panel A of the
lower group of serial plots) shows any indication of serial correlation
(positive, in this case), as may be expected given the pattern displayed by
these residuals in the lower scatter diagram of Figure 4.7.
4.7.4. Outliers
Occasionally, a residual (or residuals) may be encountered that possesses

an unusually large (absolute) value. A data point giving rise to such a
residual, termed an outlier, suggests that some error occurred during acquisi-
tion of the experimental data to generate the erroneous observation. The
presence of such a point in the data set may substantially affect the values of
derived parameters characteristic of the fitting equation used in analysis. This
possibility makes it imperative to identify such a point or points and, perhaps,
to reanalyze the data set without considering the outliers during the estima-
tion process.
The presence of potential outliers may be conveniently detected by visual

inspection of residual plots.(24) Scatter diagrams will frequently permit ready
identification of residuals which possess particularly large absolute values. A
cumulative frequency plot of residuals graphed on normal probability paper
will suggest the occurrence of outliers by the obvious presence of points which
reside distant from the majority of linearly arranged points.
Reanalyzing the data set without outliers may result in significantly
altered fitting parameter values relative to those derived with the bad points
present. A more accurate characterization of the observed experimental
phenomenon is likely to be obtained from such a reanalysis. It is sometimes
difficult, however, to have confidence that a particular point should be
rejected as an outlier rather than being retained as a point which just has a
relatively low probability of being valid. A commonly employed method for
making this decision involves rejecting any points whose residuals have
absolute values greater than 2.5 or 3 standard deviations from zero. However,
even with residuals that follow a perfect Gaussian distribution, this criterion
can sometimes incorrectly suggest the presence of outliers. For example, the
probability that any particular data point will yield a residual 3 standard
deviations from zero is about 0.0025. The probability of encountering at least
one data point with a residual 3 standard deviations from zero in a data set
consisting of n points is therefore approximately n times 0.0025. So, as n
increases, the probability of encountering a point falsely indicated to be an
outlier increases proportionately. It is expected that this criterion will result in
one point being incorrectly judged to be an outlier for approximately every
400 (or 1/0.0025) data points.
4.7.5. Influential Observations
As discussed in the previous section, outliers may cause derivation of

model parameter values that are inappropriate due to bias introduced by the
presence of a bad point in the data set being analyzed. Tests for such an
influential observation may be applied in order to determine the sensitivity of
derived parameter values to outliers.(26) It is possible that an outlier may exert
little influence on derived model parameter values if many data points are
distributed closely around the outlier in independent variable space. A point
relatively isolated from others in independent variable space, on the other
hand, may not appear as an outlier by the conventional methods of identi-
fying such points but may nonetheless be an influential observation with
regard to derived parameter values. This is a very important point! The
former case will lead to conclusions which are not substantially affected by a
bad point. The latter situation, however, will probably not identify the point
in question as an outlier but could very significantly influence the values of

derived parameters.
An observational oversight giving rise to a point which strongly influences
derived parameter values but is not detectable as an outlier presents a
unique challenge for identification. An approach in which suspected influential
observations are deleted from the data and reanalysis is performed on this
modified data set may permit identification of potential influential points
which are inconsistent with the rest of the experimental observations, given a
particular analytical model. It is possible, however, that the points deleted
almost exclusively define certain of the parameters of the analytical model.
Their exclusion may thus lead to inability to define these particular
parameters. Such a situation may seem inconclusive but actually should make
apparent to the experimenter that greater experimental determination is
necessary in this region of independent variable space, that is, that more
experimental observations are needed around these values of the independent
variable(s).
A number of quantitative protocols have been presented in the literature
to deal with situations involving influential points.(26) The details of these will
not be dealt with here. A common feature of these methods, as applied to
least-squares analysis, involves evaluating the fitting parameters from data
sets in which individual data points which are suspected influential observa-
tions are omitted. The resulting variations of these fitting parameters then
indicate the influence that a particular experimental observation has in
determining the fitting parameter values. When data points are identified as
being particularly influential, then more data should be obtained in the
neighborhood of those particular points to minimize the chances of deriving
significantly biased parameter values.
4.7.6. Common Quantitative Tests
4.7.6.1. The Chi-Square Test

Residuals that appear to follow a Gaussian distribution about zero with
no detectable trends or systematic behavior with respect to any relevant
variables, dependent or independent, may be used to test the adequacy of
an analytical model for accurately estimating observed experimental data.
A commonly employed test for this purpose involves calculation of the chi-
square statistic. (16) It can be shown that increases very nearly linearly with
the number of degrees of freedom when the analytical model is indeed correct.
Therefore, the reduced value, the value divided by the number of degrees
of freedom, will be approximately equal to one under these conditions.
A potential problem that makes implementing this test very difficult is that
estimates of the uncertainties associated with the values of the dependent

variable must be known with great confidence. These uncertainties in effect
contribute to normalization of the squared residuals such that the value
may have significance.
When reliable estimates of the uncertainty in the dependent variable are
available, a chi-square statistic is calculated as
where and are the observed and model-dependent calculated

values of the ith dependent variable, respectively, is the standard error
associated with and n is the number of data points, or dependent
variable values, contained in the data set being analyzed. The value of is
divided by the number of degrees of freedom to obtain the reduced form of
this parameter as:
where n is the number of data points, and c is the number of parameters

estimated in analysis. The value of should not be much greater than one
if the model used accurately estimates the experimental data. Statistical
probabilities that the analytical model employed is incapable of adequately
characterizing the data may be obtained by comparing the derived values of
with statistical tables of reduced values for (n – c – 1) degrees of
freedom.
An example of this analytical tool is presented in Table 4.11 as applied to
the approximately Gaussian distributed residuals of Table 4.7 and Figure 4.7.
The experimental uncertainties, presented in Table 4.11 produce the
reported values and the associated probabilities that the model used in
analysis provides a statistically valid characterization of the experimental
data. For purposes of this example, it was assumed that four parameter values
were derived from a least-squares analysis of 25 experimental observations, thus
producing 20 degrees of freedom (NDF = n – c – 1, where NDF = 20 because
n = 25 and c = 4). There would be little justification for rejecting this
(hypothetical) analytical model as long as was actually estimated to be
greater than or equal to approximately 0.74 (at a 1 % level of confidence).
4.7.6.2. The Durbin–Watson Test

The Durbin–Watson test is a test commonly employed for determining
whether a certain type of serial correlation exists in residuals.(26) The null
hypothesis is that no correlation exists in the residuals. This implies that the
residuals follow a Gaussian distribution with some constant variance, The

alternative hypothesis, in this case, is formulated as
where and are the ith and (i – l)th residuals, respectively, and and
are parameters defining the form of serial correlation hypothesized to be
present in the residuals. The are assumed to follow a Gaussian distribution
with some constant variance, and are independent of all and when
It is also assumed that the have a constant mean and variance and are
independent of when Therefore, the follow a Gaussian distribution
with the variance given by When no serial correlation is apparent,
and the situation reduces to that stated in the null hypothesis.
To decide whether or not any serial correlation is present in the residuals,
a parameter, d, is calculated as
Two critical values of and specifying a range, are involved in

deciding whether or not to reject the null hypothesis. Tables of these critical
values, at various levels of confidence, are used to decide whether

or In case (a), testing a value of implies
significance, at the level specified in the table used, and is accepted. If
then d is not significant and is rejected. If the test is
considered inconclusive. In case (b), testing the value of d above is
replaced by (4 – d), and the same criteria as in case (a) are applied. In case
(c), testing a value of or of is significant and at
twice the significance level specified in the table (because it is a two-sided
test). If and is said to be equal to zero, again at twice
the significance level of the table used. Otherwise, the test is inconclusive.
Avoiding the inconclusive cases may be approximately accomplished by
exclusively using the critical value in place of for all of the above-
mentioned comparisons. This simplified test should, however, be used only if
the original test is in the inconclusive region. The significance level associated
with the simplified test is then somewhat higher than that specified in the
table used.
This test, when applied to the residual distributions of Table 4.7 and
Figure 4.7, yields a value of d = 2.27 for the approximately Gaussian dis-
tributed residuals and a value of d = 0.93 for the non-Gaussian distributed
residuals. Tables of and values reveal that, for 25 observations and
one independent variable, (at 5% confidence), (at 2.5%
confidence), and (at 1% confidence) and that (at 5% con-
fidence), (at 2.5% confidence), and (at 1% confidence).
The conclusions from this exercise are that the approximately Gaussian
distributed residuals show no apparent serial correlation, that is, and
that the non-Gaussian distributed residuals display serial correlation with
4.7.6.3. The Kolmogorov-Smirnov Test

Testing whether residuals follow a Gaussian distribution is critical to an
evaluation of the adequacy of an analytical model for accurately estimating
observed experimental behavior. One way in which this may be accomplished
is by applying the chi-square test of goodness of fit to distributions as
described earlier. The chi-square procedure, however, requires grouping the
residuals into discrete intervals, thus discarding some details of the informa-
tion contained in the original, more continuous distribution of residuals. The
chi-square procedure also requires a minimum population in each interval in
order to permit valid characterization of all regions of the distribution. To
generate more continuous derived distributions, more data points are needed
so that a greater number of more closely spaced intervals may be used in
analysis.
The Kolmogorov–Smirnov test for goodness of fit to distributions is an
alternative to the more familiar chi-square procedure and does not suffer from
the aforementioned limitations. This test permits a statistically quantifiable
comparison to be made between a sample cumulative distribution and
some theoretical cumulative distribution function. A cumulative distribution
function is that which gives the probability that a value less than or equal to
some specified value will occur. The statistic calculated in the Kolmogorov–
Smirnov test, D, is the maximum difference between the observed and
theoretical cumulative distribution functions over all values of the variable
under consideration, X (Ref. 25, p. 387):
where and are the sample and theoretical cumulative distribution

functions, respectively, and means “the supremum (largest) over all X.”
Tables of this statistic, as a function of significance level and size of sample
(Ref. 25, p. 483), are then consulted in order to decide whether and
are statistically different at some specified confidence level.
In practice, the parameter D will be obtained by considering discrete
points of and that is, at values of i = 1, 2, ..., n. Because
is continuous with X and is discrete over finite intervals of X, both
endpoints of in each interval must be considered relative to

so as not to overlook any of the relevant distances between the sample and
theoretical functions. Therefore (Ref. 25, p. 391),
The distances between and each endpoint of each discrete portion of

are thus considered by this expression.
Figure 4.10 demonstrates the process implied by Eq. (4.69). The five points
presented in this example produce a mean value of zero and a calculated
standard deviation of one. A cumulative frequency plot is generated with
cumulative frequency (expressed as probability, i.e., ranging from zero to one)
as the ordinate (Y axis) and the value of the ordered points (in increasing
magnitude) as the abscissa (X axis). The continuous line corresponds to
the theoretical distribution function for a Gaussian distribution with a
standard deviation of one. The Kolmogorov–Smirnov D parameter for this
example has a value of 0.241, the maximum vertical distance between the
observed and theoretical cumulative probability functions. Examination of a
statistical table for this parameter indicates that there is no justification for
rejecting the hypothesis that these five points are Gaussian distributed with a
standard deviation of one at any reasonable level of confidence (Ref. 25,
p. 483).
4.8. Implementation Notes
Before proceeding to code a computer program, an investigator must first

address a simple question which has very subtle implications. The question is
“What computer language should be used for this type of analysis?” Our
answer is almost always FORTRAN-77. There are six primary considerations
which need to be addressed before the computer language is chosen. They are:
1. The computer language must be universal.
2. Only the “language standard” should be used†; i.e., no non-standard
extensions.
3. The investigator should consider the language features required for
the particular problem.
4. The computer program should be efficient.
5. Options inherent within certain languages can make the particular
problem easier.
†
A “language standard” is the internationally recognized definition of the computer language.
For example, the American National Standards Institute defined FORTRAN-77 as their X3.9-1978
standard.(28)
6. There is no need to reinvent the wheel by rewriting software which

has already been written and is available at a lower cost than that of
paying a programmer to do it again.
The optimal choice of a language is one which meets all of the investigator’s
absolute requirements and is available on almost every computer system.
For example, ten years ago when at the National Institutes of Health, one
of us (MLJ) asked their computer specialists which language should be used.
He was told that if he wanted to use their Digital Equipment Corporation
computer (DEC System 10), he should use the SAIL language, and if he wanted
to use their International Business Machines computer (IBM 360), he should
use PL/1. This investigator instead chose FORTRAN. Later, after moving
to the University of Virginia, he discovered that SAIL programs can only
run on DEC System 10/20 computers. Furthermore, the University of
Virginia’s Control Data Corporation computer (CDC 855) did not have a
PL/1 compiler. However, all three computers had FORTRAN compilers, and,
consequently, there was no need to translate any programs. Translations
can be very time-consuming and expensive, especially when the computer
programs are long and complex. Almost every computer currently in use has
a FORTRAN-77 compiler available. Most computers now also have Pascal, C,
and BASIC available. Other languages like ALGOL, BLISS, FOCAL, and Modula-2
are not commonly available. Therefore, in this discussion of relative merits we
will consider only the most popular languages: FORTRAN-77, Pascal, C, and
BASIC.
The reader is cautioned not to use the convenient language extensions
which most manufacturers have included in their compilers. For example,
some versions of BASIC allow the direct manipulation of matrices while others
and the language standard do not. Consequently, if a BASIC program which
uses these matrix features on one computer is transfered to another computer
whose compiler does not have these extensions, the program will not run.
Another example is the number of attractive services provided by the UNIX
operating system which are exclusive to particular versions of UNIX. If a
computer program is developed which relies on operating system features on
one computer, it will be difficult or impossible to transfer it to a different
computer system! To make matters more confusing, a number of computer
languages, such as Pascal and BASIC, have such poorly defined “standards”
that they vary substantially among computers from different manufacturers.
A computer program should use only those features of a language which
are the same on all computers which implement that language, that is, the
minimal language standard.
There are a number of computer language features that are required for
the development of least-squares analysis programs which are not common
among FORTRAN -77, Pascal, C, and BASIC. BASIC has a number of unattractive
features for this application. It does not always permit the easy use of sub-
routines (or subprocedures), and it does not always permit more than single-
character variable names. A potential problem with Pascal relates to the
“Pascal standard,”(27) which does not provide for double-precision floating-
point variables. Matrix manipulations, such as dot products, must be performed
with greater precision than that offered by single-precision floating-point
calculations, which typically store variables as 32-bit numbers. Furthermore,
the “Pascal standard” also does not allow subprocedures to be compiled
separately from the main program. It is inefficient and time-consuming to
recompile thousands of lines of Pascal code in order to make a simple change
in a ten-line subprocedure. The Pascal enthusiast can argue that most Pascal
compilers are not restricted by the “Pascal standard.” However, beware
that different compilers implement these features in different ways and thus
translation from one computer system to another may be difficult.
The efficiency of a computer program is important for some of the
procedures which we have presented, which require hundreds of evaluations
of complex functions. Inefficiencies can be grouped into two categories:
(a) the inherent slowness of a language, for example, BASIC, which is usually
interpreted† rather than compiled, and (b) inherent inefficiencies of some
compilers. For example, we have two different Pascal compilers for our
DEC PDP-11/73 running the operating system. We used a prime
number generation program to compare our Pascal-2 compiler with our
NBS-Pascal compiler†† and found that the NBS-Pascal compiler required
almost five times as much computer time for the same calculation. However,
the Pascal-2 compiler was substantially more expensive.
Of less importance, but still worth considering, are the optional features
which the languages do not have in common. None of these are required for
the least-squares application but are needed in some other applications. The
FORTRAN “labeled common blocks” are very useful and are not allowed by the
Pascal, C, and BASIC standards. The FORTRAN “equivalence statements” can
also be very useful. Recursion, the ability of a procedure, or subprocedure, to
†
An “interpreted” computer language is one in which code is translated to machine instructions
line by line. Each translated line is executed and discarded before the next line of the computer
program is compiled. This process is usually substantially slower than that followed in the case
of a “compiled” computer language, whereby the entire computer program is translated into
machine instructions, then executed in a separate subsequent step.
is a time-sharing operating system for DEC PDP-11 computers which is available
f r o m S and H Computer Systems, Inc., 1027 I7th Avenue South, Nashvill, Tennessee 37211.
This operating system is essentially an extension of the DEC RT-11 operating system.
††
Pascal-2 is a commercial Pascal compiler which is available from Oregon Software, 2340 S.W.
Canyon Road, Portland, Oregon 97201. NBS-Pascal is available from the Digital Equipment
Computer Users Society (DECUS), 219 Boston Post Road, Marlboro, Massachusetts 01752,
for a nominal fee. The NBS-Pascal compiler is included in the Spring 198S RT-11 Special
Interest Group Distribution Tape.
either directly or indirectly call itself, can also be useful. The and
BASIC standards do not allow recursion, but some implementations of these
languages do allow recursion. Another language feature which is attractive,
but not essential, is the ability to write “structured” programs,
and allow structured programming, while Pascal and C require struc-
tured programming. It should ne noted that it is sometimes very convenient
not to be required to use structured programs, even though it is usually an
excellent way to program.
The last important point is to avoid writing routines which have already
been programmed. Subroutine libraries exist to perform most of the linear
algebra and eigensystem operations required for least-squares parameter
estimation. Examples are the and
libraries of subroutines. These are all written in and/or
Some computers will allow linking a routine written in one
language to a program written in another language if certain specific condi-
tions are met, but this is a difficult process at best. A better way to approach
the encoding of an analysis procedure is to begin coding in the language of
choice, that is, the standard, so that implementing preexisting
routines to handle complicated, but standard operations is straightforward.
There is little justification for ever converting a functional computer program
from one language to another unless the computer system being used does not
have a compiler for the first language. Again, we recommend that computer
programs be written in either the standard (28) or, sometimes,
if the computer is running the operating system, the C language
standard. (29)
4.9. In Summary
Computers are not oracles. The results of any computer analysis are
dependent on the quality of the data, on the assumptions of the analysis
method, and on the expertise of the computer programmer who generated the
software. Consequently, computer users should always be aware that com-
puter programs might give them wrong answers. Computer programs should
always be tested with various types of “test” data and the possibility that
answers derived from the analysis of actual experimental data may have no
physical meaning should always be considered.
Two of the cited references are excellent entry-level numerical methods
and comprise a library of subroutines to perform least-squares function

minimization and parameter estimation problems. They are available from the International
Mathematical and Statistical Library (IMSL) Distribution Service, P.O. Box 4605, Houston,
Texas 77210-4605, for a nominal charge.
textbooks.(7,16) Explicit definitions of several computer languages are provided

in the following sources: FORTRAN -77 in Ref. 28, C in Ref. 29, and Pascal in
Ref. 27. In addition, several of the references provide subroutine libraries
printed in the text. (7–9, 11,16) Some subroutine libraries can be obtained on
magnetic media for a nominal fee. (8,9,1l)
The nonlinear least-squares procedures presented in this chapter have a
sound statistical basis if certain assumptions are met. A number of authors
have questioned the validity of the assumptions and, as a consequence, of
least-squares procedures (3) However, no other procedure has as good a
basis in statistics as least-squares, which is a special case of the more general
maximum-likelihood method. As long as the assumptions are reasonable,
least-squares analysis is the best available method because it yields answers
which have the highest probability of being correct.
References
1. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, pp. 65–82, Plenum Press, New York
(1983).
2. M. L. Johnson, The analysis of ligand binding data with experimental uncertainties in the
independent variables, Anal. Biochem. 148, 471–478 (1985).
3. I. Isenberg, Robust estimation in pulse fluorometry: A study of the method of moments and
least squares, Biophys. J. 43, 141 (1983).
4. N. Joshi, M. L. Johnson, I. Gryczynski, and J. Lakowicz, Radiation boundary conditions in
collisional quenching of fluorescence: determination by frequency domain fluorometry, Chem.
Phys. Lett. 13S, 200–207 (1987).
5. M. L. Johnson and S. G. Frasier, Nonlinear least-squares analysis, Methods Enzymol. 117,
301–342 (1985).
6. J. A. Nelder and R. Mead, A simplex method for function minimization, Comput. J. 7,
308–313 (1965).
7. G. E. Forsythe, M. A. Malcolm, and C. B. Moler, Computer Methods for Mathematical
Computations, Prentice-Hall, Englewood Cliffs, New Jersey (1977).
8. B. T. Smith, J. M. Boyle, J. J. Dongarra, B. S. Garbow, Y. Ikebe, V. C. Klema, and
C. B. Moler, in: Matrix Eigensystem Routines—EISPACK Guide, Second Ed. (G. Goos and
J. Hartmanis, eds.), Springer-Verlag, New York (1976).
9. B. S. Garbow, J. M. Boyle, J. J. Dongarra, and C. B. Moler, in: Matrix Eigensystem
Routines—EISPACK Guide Extensions, (G. Goos and J. Hartmanis, eds.), p. 69, Springer-
Verlag, New York (1977).
10. V. N. Faddeeva, Computational Methods of Linear Algebra, p. 81, Dover, New York (1959).
11. J. J. Dongarra, C. B. Moler, J. R. Bunch, and G. W. Stewart, LINPACK Users' Guide, Society
for Industrial and Applied Mathematics, Philadelphia (1979).
12. F. B. Hildebrand, Introduction to Numerical Analysis, p. 82, McGraw-Hill, New York (1956).
13. M. S. Caceci and W. P. Cacheris, Fitting curves to data, Byte Magazine 9(5), 340–362
(1984).
14. W. Spendley, G. R. Hext, and F. R. Himsworth, Sequential application of simplex designs in
optimization and evolutionary operation, Technometrics 4, 441 (1962).
15. C. Hastings, J. T. Hayward, and J. P. Wong, Approximations for Digital Computers, p. 187,
Princeton University Press, Princeton, New Jersey (1955).
240 Martin Straume et at.
16. P. R. Bevington, Data Reduction and Error Analysis for the Physical Sciences, pp. 187–203,
McGraw-Hill, New York (1969).
17. G. E. P. Box, W. G. Hunter, and J. S. Hunter, Statistics for Experimenters, p. 636, Wiley-
Interscience, New York (1978).
18. M. Zelen, and N. C. Severo, Probability functions, in: Handbook of Mathematical Functions
with Formulas, Graphs, and Mathematical Tables, Ninth Printing (M. Abramowitz and
I. A. Stegun, eds.), pp. 925–997, National Bureau of Standards Applied Mathematics Series
#55, Washington, D. C. (1970).
19. G. E. P. Box, Fitting empirical data, Ann. N.Y. Acad, Sci. 86, 792–816 (1960).
20. M. L. Johnson, H. R. Halvorson, and G. K.. Ackers, Oxygenation-linked subunit interactions
in human hemoglobin: Analysis of the linkage functions for constituent energy terms,
Biochemistry 25, 5363–5371 (1976).
21. M. L. Johnson, J. J. Correia, D. A. Yphantis, and H. R. Halvorson, Analysis of data from the
analytical ultracentrifuge by nonlinear least-squares techniques, Biophys. J. 36, 575-588
(1981).
22. M. L. Johnson, Evaluation and propagation of confidence intervals in nonlinear, asymme-
trical variance spaces: Analysis of ligand binding data, Biophys. J. 44, 101–106 (1983).
23. P. Armitage, Statistical Methods in Medical Research, Fourth Printing, pp. 316–319,
Blackwell Scientific Publications, Oxford (1977).
24. Y. Bard, Nonlinear Parameter Estimation, pp. 201–202, Academic Press, New York (1974).
25. W. W. Daniel, Biostatistics: A Foundation for Analysis in the Health Science, Second Ed.,
John Wiley & Sons, New York (1978).
26. N. R. Draper and H. Smith, Applied Regression Analysis, Second Ed., pp. 153–174,
John Wiley & Sons, New York (1981).
27. K. Jensen and N. Wirth, Pascal User Manual and Report, Second Ed., Springer-Verlag,
New York (1978).
28. L. P. Meissner and E. I. Organick, FORTRAN 77: Featuring Structured Programming,
pp. 427–482, Addison-Wesley, Reading, Massachusetts (1980).
29. B. W. Kernighan and D. M. Ritchie, The C Programming Language, Prentice-Hall,
Englewood Cliffs, New Jersey (1978).
5
The Global Analysis

of Fluorescence Intensity and
Anisotropy Decay Data:
Second-Generation Theory
and Programs
Joseph M. Beechem, Enrico Gratton, Marcel Ameloot,
Jay R. Knutson, and Ludwig Brand
5.1. Introduction
Time-resolved fluorescence spectroscopy has proven to be a powerful

physical technique for the studies of fast reactions and dynamics on the sub-
picosecond to microsecond time scales. Examination of the decay kinetics of
specific excited state photophysical processes (e.g., resonance energy transfer,
solvent relaxation, excimer/exciplex formation, dynamic quenching, proton
transfer, rotational diffusion, etc.) can provide important information concer-
ning reactions approaching equilibrium. When the fluorescent molecule is an
integral part of a larger biological system (e.g., proteins, membranes, macro-
molecular structures), the decay kinetics can reveal information concerning
the structural dynamics of these complicated systems.
There have been significant advances in the instrumentation used to
Joseph M. Beechem and Enrico Gratton • Department of Physics, University of Illinois

Urbana–Champaign, Urbana, Illinois 61801. Marcel Ameloot • Limburgs Universitair
Centrum, Universitaire Campus, B-3610 Diepenbeek, Belgium. Jay R. Knutson • National
Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Ludwig Brand • Department of Biology, The Johns Hopkins University, Baltimore,
Maryland 21218. Present address for J.M.B.: Department of Molecular Physiology and
Biophysics, Vanderbilt University, Nashville, Tennessee 37232. Information concerning a
commercial version of the program described in this chapter can be obtained by writing to:
Globals Unlimited, Laboratory for Fluorescence Dynamics, University of Illinois, Urbana,
Illinois 61801. No endorsement by the U.S. Government is implied.
241
242 Joseph M. Beechem et al.
obtain time-resolved fluorescence data during the past ten years. The use of
high-repetition-rate picosecond dye lasers and fast detectors (such as micro-
channel plate photomultipliers) has had a major impact on both time- and
frequency-domain measurements. Data acquisition time has been greatly
decreased, especially for single-photon counting experiments, which used to be
limited by the repetition rate of the excitation light source. Accurate single-
photon counting decay data may now be collected in under a second(1–3) and
will probably approach the millisecond time scales very soon. These fast
collection rates are beginning to make it possible to perform “double-kinetic”
experiments: multiple picosecond-microsecond fluorescence experiments per-
formed on the millesecond time scale. Reports of similar efforts to decrease
data acquisition time in the frequency domain have also appeared.(4) Parallel
(“multifrequency”) data collection(5) (i.e., where many frequencies are acquired
simultaneously) should soon result in a drastic reduction in data acquisition
time in the frequency domain. Alternative stroboscopic data acquisition
schemes in the time domain are also being developed.(6)
Many numerical techniques, such as nonlinear least squares,(7–9) method
of moments,(10,11) Laplace transforms, (12,13) and modulating functions, (14)
have been used to analyze fluorescence decay data. It has been found that
for the accurate recovery of closely spaced exponentials (or distributions
of exponentials), the analysis of individual fluorescence decay experiments
is usually insufficient. For the accurate recovery of complex fluorescence
decay phenomena, it is advantageous to combine more than one fluorescence
decay experiment into a single analysis.(15–20) A number of nonlinear last-
squares(19–26) and Laplace transform (27) software routines have been developed
with multiexperiment capabilities. The simultaneous analysis of multiple
fluorescence decay experiments is frequently referred to as “global” analysis
and has proven useful for both time- and frequency-domain data.
Global analysis procedures are of significant advantage when some
unknown parameter(s) of interest is linked between two or more fluorescence
decay experiments obtained under different conditions. A “condition” in this
context refers to temperature, pressure, excitation/emission wavelengths, or
any other independent variable which can be experimentally manipulated. In
many situations, the parameters are not linked directly between experiments,
but rather through a mathematical function. The overall strategy behind a
typical global analysis can best be illustrated with a few examples.
5.1.1. Multiexcitation/Multitemperature Studies of Anisotropic Rotation
For nonspherical molecules, the decay of the emission anisotropy is

described by a sum of exponentials † with rotational correlation times inde-
†
For cases of depolarization due to rotational diffusion.
Global Analysis of Fluorescence Intensity and Anisotropy Decay Data 243
pendent of the excitation wavelength and preexponential terms dependent on

the relative direction of the absorption/emission oscillators with the principal
diffusion axes of the molecule.(28) In this case, global analysis of experiments
done at several excitation wavelengths provides a useful overdetermination
of the rotational correlation times. The multiple excitation experiments
are analyzed for an internally consistent set of correlation times, whose
preexponential factors are allowed to vary as a function of the excitation
wavelength (example of a simple global “linking” of the rotational correlation
times over the multiple excitation wavelengths).
Experiments can also be performed at multiple temperatures (T) and/or
viscosities The rotational correlation times cannot be directly linked
between these experiments, because they change with T and To link rota-
tional rates over multiple temperatures and viscosities requires a mathe-
matical function which predicts how these quantities will change. Many simple
molecules in solution show classic Stokes–Einstein type of behavior over
particular ranges. In these cases, one can link rotational parameters over
temperature using this relationship. This allows one to explore those regions
where the lifetime may be much faster/slower than the rotational relaxation
rate.
5.1.2. Multiexcitation/Emission Wavelength Studies of Total Intensity Data
Global analysis has also proven useful in the evaluation of a mixture of

two or more noninteracting fluorescent species, each with a unique decay
function and excitation/emission spectrum. Global analysis of a data set
obtained as a function of emission wavelength, with linkage of decay con-
stants, allows more closely spaced lifetimes to be recovered than single-curve
analysis alone. In addition, the preexponential terms may be used to recover
the individual spectra associated with the particular decay time (decay-
associated spectra, DAS). (29,30,17) The occurrence of discrete DAS for
individual tryptophan/tyrosine residues in many different proteins(31) is
proving to be useful for monitoring structural changes occurring in different
regions within a single protein molecule in solution. These DAS have also
proven to be useful in resolving macromolecular interactions and sitespecific
quenching. (l,32) The assignment of specific spectra to different fluorescence
lifetimes may be further aided by utilizing additional independent variables,
such as quencher concentration. If DAS can be related to a specific tryp-
tophan residue (or conformation), then one might expect that different
dynamic quenching constants may be "linked" to each spectrum (quenching-
decay associated spectra, QDAS). Indeed, spectra associated with distinct
quenching constants have been recovered and can be obtained by either
global or nonglobal methods; usually, both are combined.(1,33–35) Just as for
lifetimes, closely spaced quenching decay constants are more easily resolved
by global multiwavelength analysis (either steady state or time-resolved).
5.1.3. Double-Kinetic Studies
Another example where global analysis has been helpful is in studies of

the kinetics of protein unfolding/refolding on the second to minute time
scales.(1–3) Complete fluorescence decay curves were collected every few
seconds, and the effects of unfolding on the lifetime component(s) were
observed. The individual decay curves are too noisy to support complex
analysis. However, when the entire data surface (collected during the protein
conformational change) is globally analyzed using various linkage schemes,
time-dependent changes in lifetimes and amplitudes can be recovered. In this
manner, a direct correlation of the picosecond/nanosecond fluorescence decay
kinetics with macroscopic millisecond/kilosecond biological reactions is made
possible.
5.2. The Global Analysis Philosophy
5.2.1. Evolution of the Global Analysis Approach
The global analysis examples described above (and many others) have
proven very useful for many different studies. However, these examples mainly
represent an adaptation of classical single-curve analysis algorithms to perform
a global analysis. The global analysis approach has since been expanded, in
a manner that is farther removed from simple curve fitting, to an emphasis on
physical-model evaluation.
For instance, in the rotational diffusion global analysis example (see Sec-
tion 5.1.1), the quantities of physical interest are not the preexponential factors
and characteristic rotational correlation times, but rather the shape and size
of the molecule and its interactions with the solvent. There is ample theory
which describes the relationships between the size and shape of molecules and
the exponential relaxation times which are observed. An important result
which often arises from theory is the fact that the observed set of rotational
correlation time(s) and preexponential factor(s) are in general not
independent of each other. If the data analysis is performed in space,
then the recovered values for these parameters may mathematically represent
the data very well, but may not have any physical analogue. Abondoning this
type of curve fitting and directly analyzing the rotational data in terms
of the physical parameters of the system has been termed “target analysis”
( physical model fitting) by Arcioni and Zannoni(36) and has been rigorously
adopted by the current global analysis programs. See Ref. 37 for a recent
study of anisotropic rotations using a “global-target” analysis of differential
phase/modulation data obtained at multiple temperatures.
In the current global analysis programs, every effort has been put forth
to allow the user to select the physical invariants of the system as fitting
parameters, obviating multistep analyses. In the case of rotational diffusion,
the physical invariants of the system are the lengths of the major axes of the
molecule, the set of angles which describe the orientation of the absorption/
emission oscillators and the principal diffusion axes of the molecule, and the
“slip” or “sticking” boundary conditions. Of course, from a single anisotropy
experiment, one should not expect to recover all the values of the physical
invariants of a system, and, generally, a global analysis proceeds using the
following approach.
Initially, the emission anisotropy may be fit to a series of exponential
terms. From these results, some of the possible physical models for the
molecular shape can be disregarded. For example, if the data fit very well to
only a single rotational correlation time, then there is little reason to perform
an analysis in terms of lengths and multiple diffusion coefficients. However,
upon excitation of the molecule into different absorption oscillators, it may be
found that the single recovered rotational correlation time varies. One should
then consider performing a global analysis of the two data sets in terms of an
internally consistent molecular shape with varying angles describing the
relative orientation of the absorption and emission oscillators and the principal
diffusion axes of the molecule.
Experiments performed at multiple temperatures and viscosities can
further assist in unraveling complex decay. In this case, one needs to utilize
a physical model which will relate how these variables affect rotational
behavior. For instance, one can model rotational diffusion as an activated
process and directly analyze the data in terms of the energy of activation for
rotational diffusion (i.e., the physical invariant of the system).
From this example, a general pattern for performing a global analysis
emerges:
1. Examine the individual data sets in a semiempirical manner (e.g.,

using sums of exponentials, parameterized or maximum–entropy
lifetime distributions). Utilize this information as a preliminary step
in establishing a set of possible physical models to be applied to the
system.
2. Change the fitting parameter space from the semiempirical fitting
parameters (amplitudes and relaxation times) to more “target”
parameters, which represent the physical invariants of the system.
3. In order for this “target” fitting space to be useful, one requires a

mapping of the physical model that has been chosen to the space in
which the experimental data have been collected. This mapping
scheme represents the section(s) of code which embody, in an algo-
rithmic fashion, the theory of this particular physical model (e.g.,
compartmental modeling of pholophysical reactions, partial differen-
tial diffusion equation solver for energy transfer, etc.).
4. Once the physical model has been completely specified (i.e., the
physical invariants of the system, relationships to the experimental
observables), one can perform a global analysis (parameter optimiza-
tion) of the multidimensional data surface. In this type of analysis, all
of the implicit linkages which exist between the fitting parameters and
the data sets are utilized.
5. Rigorous error analysis† is now applied to the recovered parameters

of the model.
6. Alternative physical models are applied to the data surface.
This process is schematically represented in Fig. 5.1.
Often, it is found that the global analysis error surface is much better
defined than the individual curve error surfaces (item 5 above; see Figure 5.2).
Therefore, the fitting parameters can be recovered with much smaller uncer-
tainties than in individual curve analysis. This is often proclaimed as the
major advantage of global analysis. Actually, an equally important aspect of
global analysis is its model-testing capability.
It may be desirable to apply several different physical models to a
particular data surface (item 6 above). In this case, a whole series of physical
models, each with its own distinct set of linkages, would be applied to the
data. The global analysis error statistics can now be invoked to “rank”
the possible sets of models as to how well they represent the experimental
observables (see Figure 5.1).
It is consistently found that both parameter recovery and model-testing
capabilities are enhanced upon performing global analysis. As will be
†
To be described in Section 5.7.3.
described in more detail in subsequent sections, the increased discrimination

capability arises from the drastic reduction in the number of fitting parameters
obtained throughout the global model specification process.
It should be emphasized that global analysis is in no way limited to
one type of experimental data. Fluorescence decay may be combined with
steady-state fluorescence, absorbance, or other types of experimental data.
The only requirement is that the equations representing the various types of
experiments have some common terms as fitting parameters.
5.2.2. Global Analysis Implementation Strategy
There are three basic methods with which one can implement a global
analysis program:
1. Specific case programs: If the number and type of experiments which
are going to be examined are relatively limited, one may wish to
directly hardwire specific global linkages directly into the software.
For instance, combining multiple excitation/emission wavelengths in
terms of an internally consistent set of lifetimes is a specific case, and
several special-purpose routines have been written for these types of
experiments.
2. User subroutines: Nonlinear minimization routines can be written
which only contain the logic needed to perform a global minimization
over multiple experiments, but there is no explicit linking mechanism
within the analysis program. In this case, each particular user (of
the program) would be required to write a subroutine, which would
contain all of the logic required to link the various experiments
together.
3. General-purpose linking algorithms: In a general-purpose linking
nonlinear least-squares algorithm, a mechanism within the program
allows the user to explicitly enter the number and types of linkages
desired over the entire data surface. A general-purpose numerical
algorithm is utilized to calculate the observed fluorescence response;
all derivatives are calculated numerically, so that a wide variety of
complex models can be examined.
Of the above-described methods, no single methodology is advantageous
in all cases. Specific-case algorithms suffer from a lack of flexibility, but are
often very fast and produce relatively compact programs. This type of
methodology should definitely be pursued if only a single type of global
analysis needs to be performed. Programs which rely on external user-written
subroutines are more useful, but incur the added expense of requiring that
the program be recompiled for each new type of analysis. The user must
also supply the necessary linking logic, which usually requires a reasonably
sophisticated understanding of the structure of the main program. This

methodology allows a set of subroutines to be developed which can analyze
a wide variety of data. These type of algorithms have an advantage over
general-purpose algorithms, because a large region of code and data space
devoted to all of the other models is not wasted (in memory) for the analysis
of one particular special case. General-purpose linking algorithms provide a set
of indirect addressing matrices (which are user-defined at run lime), which
completely specify both the set of fitting functions to be used and the set of
linkages desired between the multiple experiments. The major advantage of
this technique is that very complicated sets of fitting functions and linkage
types can be used in a single program.
The original set of global analysis routines† (19,20) were specifically designed
to link fluorescence lifetimes as a function of excitation/emission wavelengths.
Analysis programs were then developed for excited state reactions(24,27)
and anisotropic rotations. (23,25) Each of these programs was specific to a par-
ticular photophysical model and data structure. These programs have since
been enhanced, so that all of these models (and many more) can be examined
within a single global data analysis environment, (26) and constitute the set of
programs currently operating at the Laboratory for Fluorescence Dynamics
(LFD). These algorithms are of the general-purpose linking type and will be
described in detail below.
Although both transform techniques and standard nonlinear minimization
routines can be adapted to perform a global analysis, the nonlinear least-
squares algorithms are far easier to implement and are flexible. In this
chapter, only the nonlinear minimization algorithms will be described. The
emphasis in this chapter will not be on explicit technique for solving the least-
squares equations; the global analysis methodology can be implemented using
many different minimization algorithms. Instead, emphasis will be placed on
the “mechanics” of developing a multidimensional global analysis and on
specific examples of overdetermination which are important for fluorescence
experiments.
5.3. General Elements of the Global Analysis Program

The general scheme of the global analysis program as implemented at the
LFD is shown in Figure 5.3. The general elements of the program are as
follows:
• For the construction of the model matrix (Figure 5.3), the present
program implements both direct physical models and empirical
† No attempt has been made to reference all the laboratories which have developed a set of global
analysis algorithms. Instead, only the algorithms with which the authors have had personal
involvement are described.
models. The selection consists of forming a list of keywords that will

be later interpreted and bound to specific analytical functions. See
Section 5.4 for a full list of the keywords currently used by the present
program (this list is constantly expanding). Finally, for the construc-
tion of the model matrix, there is a possibility to specify other elements
which represent specific experimental conditions, such as the fraction
of photons absorbed for each species, current emission wavelength
settings, etc. In this manner, a physical model is completely specified
by combinations of keywords which fully describe the parameters of
the model. A supervisory editor monitors the input of the model for
self-consistency. The parameter descriptor list contains limits for the
numerical values of the various fitting parameters (e.g., the program

“knows” that distances, rates, spectra, must be positive, etc.).
• The linking matrix (Figure 5.3) is built using a list of pointers. A
graphics-based linking mechanism allows parameters of complemen-
tary type to be combined over multiple experiments. Once the physical
model has been built, the linking process consists only in the specifica-
tion of the invariants of the system.
• The data matrix (Figure 5.3) is constructed by specifying which data
files are to be used for a specific analysis. The number of elements of
the data matrix depends on the experiment to be analyzed, the number
of data points for each data curve, and the type of data examined.
• Once the three basic structures (described above) have been constructed,
the global analysis program proceeds as follows (see Figure 5.4):
• The keyword descriptor for each fitting parameter for this experi-
ment is read.
• A “parser–decoder” routine recognizes the keywords of the list and
loads the appropriate functions.
• The fluorescence intensity is calculated for all the frequencies (or
times) of a given experiment.
• Each experiment is processed in the above manner, until all of the

data surface has been examined.
• A nonlinear minimization algorithm modifies the values of the fitting
parameter to best fit the experimental data.
In the next section we illustrate (in detail) some of the most commonly
used models and algorithms for the calculation of the fluorescence intensity
(empirical sums-of-exponentials and compartmental models). We also provide
an overview of the minimization method utilized by the global analysis.
5.3.1. Mapping to the Physical Observables
In any fitting procedure, one needs to be able to calculate the experi-

mental observables, given a set of fitting parameters. This process will transfer
the parameters of the model into the decay-associated parameters. The current
program has a choice of methodologies available to perform this mapping.
This first method to be described (which is also the most commonly used) is
based upon the methods of compartmental analysis and systems theory. There
is a very large number of models and processes which can be examined using
this methodology (e.g., empirical multiexponential decay, classical excited
state reactions, some energy transfer processes, dipolar relaxations, rotational
motions, collisional and static quenching, etc.). There are, of course, systems
that cannot be represented using this mapping procedure, and alternative
methodologies must be utilized. For instance, in the calculation of the
fluorescence observables for transient effects in energy transfer and quenching,
one can utilize the numerical solution of a set of second-order parabolic
partial differential equations (an example will be described in Section 5.5.2).
What should be kept in mind, however, is that the global analysis metho-
dology is not limited in any way by the particular numerical procedure
utilized to calculate the observed fluorescence.
5.3.2. Empirical Description of the Fluorescence Decay
All the information content in a single fluorescence decay experiment

(performed in the linear regime) is contained in the impulse response function
(time domain) or the transfer function (frequency domain) of the sample. The
impulse response function and transfer function are directly related to each
other through the Fourier transform. For instance, if the time-domain impulse
response function is being modeled as a sum of exponentials, one has
The frequency-domain transfer function in complex notation (i.e., the Fourier

transform of Eq. 5.1) is
where (v is the frequency of the excitation source). In the time

domain, the observed experimental quantities are the impulse response
convolved with an excitation profile l(t):
In the frequency domain, the experimental observables are the phase

and demodulation ( M ) of the fluorescence emission with respect to the
excitation source:
where Im is the imaginary part of and Re is the real part of

Therefore, it would appear that the most natural set of fitting parameters
for the analysis of fluorescence experiments would be the elements of the
impulse response function or transfer function of the sample. However, as will
be shown, confining the fitting process to and space may be inadequate
and is often an undesirable method of analysts (taken alone).
5.3.3. Compartmental Description of Photophysical Events
The use of systems theory for the analysis and interpretation of

fluorescence decay data was pioneered by Eisenfeld and Ford.(15) This
methodology is very general, and flexible, and is implemented in the current
global analysis program. A diagrammatic representation of a fluorescence
decay experiment in terms of a systems theory view of a photophysical system
is presented in Figure 5.5. The excitation light source enters a
fluorescence cuvette which contains a mixture of ground state species. The
light partitions into the various ground state species depending upon their
concentrations and extinction coefficients. This ground state absorbance for
the ith species is denoted as A population of n excited fluorescence species,
is now produced. The interaction of the excited molecules can be
modeled using a wide variety of user-definable functions, fij.
In Figure 5.5, only a sequentially linked set of n different excited state
species (or compartments) are depicted, but any looped or branching variation
of this diagram is also allowed. The excited state species may emit fluorescence
with a functional form depicted as These emitted photons are observed by
a detector through an emission monochromator/filter which operates as a
“gain-discrimination” device, weighting the photon from each particular
species by a factor proportional to the emission spectra of that state. These
gain factors are denoted as The fluorescence actually detected will then
simply be the weighted sum of the individual emissions.
Consider the case of a simple two-state excited state reaction. These
schemes have been extensively utilized to model excited state proton transfer,
exciplex and excimer formation, etc. Historically, these types of systems have
been analyzed utilizing the simple solution to the coupled two-compartment
linear differential equations, which yields
Naturally, the individual and are functions of all the rate constants of the
system and the initial boundary conditions. Previously, systems of this type
were first analyzed in terms of individual and as a function of pH,
concentration of species, etc. These results were then analyzed graphically, to
obtain the rate constants of the system (and spectra).
With the systems theory formulation, this sequential type of analysis is
no longer needed. The kinetic system described above is fit directly in terms
of the rate constants and spectra associated with each particular system. For
this simple case, analysis with either methodology may yield comparable
results. However, in more complicated systems (e.g., many more states,
distributed or time-dependent rate constants, etc.), the analytical forms of the
impulse response functions may be very difficult (impossible?) to obtain, and,
hence, analysis using impulse response equations [e.g., Eqs. (5.6) and (5.7)]
is not possible.
The kinetics of systems shown in Figure 5.5 can be written in terms of the
standard systems theory equations as(43, 44)
Here, X(t) is the (n x 1) vector of the concentrations of each excited state

species; X(t) is the time derivative of X(t); T is the (n x n) transfer matrix
which describes the connectivity between different compartments, with
and for where fji is the
function being used to describe the transfer of photons (molecules/species)
from compartment i to compartment j; is the parameter vector which
characterizes the function associated with the transfer between (or into)
compartments; a is a vector of the zero-time absorbances of each ground state
species; and l(t) is the measured excitation function.
In a fluorescence experiment one does not directly observe the decay of
each excited state species; instead, the observed emission is weighted by the
spectral contours of each particular state. The emission spectral dependence
can be added to the “discrete-case” solution of Eq. (5.8) to yield the predicted
time course of the total-intensity (i.e., nonpolarized) fluorescence:
where is the jth eigenvalue of the matrix T (Eq. 5.8), V is the matrix of
eigenvectors of T (modal matrix), n is the number of excited state species, and
is the emission spectral contour of the kth species at the ith emission
wavelength.
Note that the final amplitude associated with a particular decay rate
(commonly denoted as is actually formed as an inner product of the “s”
vector and the eigenvector associated with that particular eigenvalue. In the
current global analysis programs, the individual can be actual fitting
parameters. The are referred to as because they represent a
particular chemical’s “species-associated spectrum” (i.e., the emission spectrum
of this species if it could be observed separately). This is in contrast to
(decay-associated spectra), a term which has come to represent the-
set of amplitudes associated with a particular decay time. † In the case of strict
ground state heterogeneity, the eigenvectors are just the normal basis vectors,
and therefore the amplitude associated with a particular lifetime is directly
proportional to its emission spectrum (i.e., for ground state heterogeneity,
DAS = SAS). However, for excited state reactions, the final observed
amplitude is a linear combination of the SAS and each particular eigenvector
(Eq. 5.9) and hence the DAS are not the same as the SAS. The advantage of
being able to fit in an “SAS” space is that, since the represent species
emission spectra (a physical invariant), they can be linked over experiments
where the kinetics of interconversion have been altered (e.g., by changing pH,
concentration of quenchers, etc.) whereas simple amplitude (DAS) terms
cannot.
The “distributed-case” solution to Eq. (5.8) [i.e., where the parameter
vector is distributed according to can be written as
In the frequency domain one has
These equations can be rewritten in the more common form
Note that the final represent the “end result” of a complex set of
operations: the inner product of the eigenvectors of and the emission
spectral contours of each state (Eq. 5.9) weighted by the multidimensional
†
Knutson et al.(30) originally defined DAS to encompass both ground state and excited state
systems.
probability function Although Eqs. (5.12) and (5.13) are of the correct
mathematical form for fitting fluorescence data, Eqs. (5.10) (convoluted) and
(5.11) are the form actually utilized in the global analysis because it allows
one to “link” specific subsets of the “lumped” parameters between
experiments.
In the case of excited state reactions, the set of all and
are not independent of each other (due to eigenvector–eigenvalue system-
wide relationships) so that these distributed cases are very difficult (if not
impossible) to solve analytically, and a numerical integration procedure must
be performed. However, for cases where there are no excited state interactions
(i.e., the T matrix is diagonal), then the complete set of and
decouple, and one can rewrite Eqs. (5.12) and (5.13) as
The integrals given by Eqs. (5.14) and (5.15) need to be calculated only over
the parameters used to characterize each individual distribution, because they
are not a function of the entire set of constants and distributions describing
the total kinetic system
Equation (5.14) is the definition of the Laplace transform of the
distribution function, Within the context of fluorescence decay experi-
ments, this integral is also similar to what is known in various other fields
as the “moment-generating function” or “characteristic function” of the
distribution. (46) Since there exist large tables of Laplace transforms, moment-
generating functions, and characteristic functions, a wide variety of analytical
forms now exist to analyze time- and frequency-domain data given a specific
analytical form for the distribution function (in addition to standard sums-of-
exponentials models).
An approach which can be taken to analyze fluorescence decay data
is to perform an inverse Laplace transform of the data to directly recover
the probability distribution associated with each lifetime. Many different
“regularized” solutions of the inverse transform have been applied with varying
amounts of success.(47, 48) In general, an entire family of distributions will be
consistent (i.e., have the same value) for any given data set. To choose
among these different distributions, one can analyze for the distribution
consistent with the data that has the largest “entropy.”(49–5l) An important
advantage of these techniques is that no analytical form needs to be assumed
to describe the distribution function itself. However, an important point to
consider is the fact that the intrinsic distribution of the amplitudes as a
function of lifetime generally does not represent the final parameters of interest
in a global analysis. These methods do not currently take advantage of multiple
axes of overdetermination.
For example, consider the case in which a particular fluorescence sample
is measured as a function of some independent variable and the distribution
of versus versus independent variable is recovered perfectly. Is this result,
in itself, of any use without a particular model which can predict such shapes?
For this reason, regardless of which analysis methodology is employed to
recover a set of versus eventually it is desirable to decompose the
recovered versus in terms of an internally consistent physical model.
It is advantageous not to operate on the versus themselves, but rather
to examine the original data surface in terms of an internally consistent
physical model. The amplitudes and lifetimes should be utilized as a guide to
performing physical modeling.
5.3.4. Overview of Nonlinear Minimization (The Basic Equations)
Nonlinear least squares involves the minimization of the chi-square

function:
where N is the number of data points in the entire data surface, is the
standard deviation of the ith data point in the qth experiment, m is the
number of total fitting parameters, is the total number of experiments,
and n(q) is the total number of data points taken in the qth experiment.
Division by the degrees of freedom (N — m—1) does not vary during each fit,
so that minimization of a nonreduced will produce exactly the same result.
The reduction process, however, allows for some preliminary model testing.
Using the definition of and assuming a Gaussian error distribution, one
may assemble F tests (or related criteria) to judge the “quality” of the fit. A
reduced that significantly differs from unity calls into question the validity
of either the model or the weighting estimates In the time domain, one
can use single-photon statistics and show that the variance in each channel
is directly proportional to the number of counts. In the frequency domain,
no simple relationships exist to predict analytically what the error in the
measured phase and modulation should be. Instead, measurements are
repeated and an experimental variance is determined. Improperly weighted
data can have drastic effects on the final recovered parameters, especially for
anisotropy decay.
The mechanics of minimization are many and varied. We have adopted
a popular and demonstrably reliable method attributed to Marquardt (40) and

Levenberg.(41) Given an initial set of guesses in a vector (the superscript
denotes the current iteration number), one obtains by forming:
The parameter improvement vector is determined from the shape of the

hypersurface around the current fitting parameters, by solving the following
linearized system of equations:
where
and is the parameter increment in fitting parameter k. In Eq. (5.19), is a

scaling factor, and I is the identity matrix. The diagonal scaling matrix
determines the “mode” in which the Marquardt–Levenberg algorithm
operates. Changing by decreasing it after each “successful” ( reducing)
step and increasing it upon step failures represents a strategy that guarantees
finding a minimum. The possibility of local minima is not obviated, however,
so the analyst must remain skeptical and exhaust alternative minimization
pathways (multiple analyses employing different initial guesses, rigorous error
analysis, etc.).
5.4. In-Depth Flow Chart of a General-Purpose Global Analysis Program
In this section, technical details concerning the implementation of the

general-purpose global analysis routine will be presented. The information in
this section should be sufficient for independent laboratories to develop their
own specific global analysis programs (if desired).
5.4.1. Overview of the Global Analysis Procedure
In relation to Figure 5.3, there are three structures which must be estab-
lished before the global optimization can be performed:
• The physical model/solution process/experimental conditions matrix
• The linking connectivity matrix
• The data matrix.
The first task in any analysis procedure is to construct the calculated

data associated with the ith experiment. In single-curve analysis, there is
only one experiment (and hence one fitting function), so that one does not
have to perform any specific parameter indexing (i.e., every fitting parameter
in single-experiment analysis is either directly used or is held as a fixed
constant). However, for a global analysis, many different experiments need
to be combined into a single analysis. Therefore, certain specific indexing
methodologies need to be added to the fitting routines so that the correct
fitting parameters and fitting functions can be obtained for each individual
experiment.
A global analysis algorithm must first recognize those elements of the
total fitting parameter vector which are used for that particular experiment.
Then it must pass these particular elements to the fitting function appropriate
for the experiment. The calculated data values for this experiment can then be
obtained. By “looping” in this fashion over the entire set of experiments being
examined, a global analysis can be performed.
A description of the logic implemented in the current global analysis
programs will now be presented. It should be emphasized that the following
steps could be implemented in many different ways. We have chosen the
following implementation because it has proven useful for fluorescence decay
data and because it can be readily adapted for the analysis of many different
types of models.
5.4.2. Flow Chart for the LFD Global Analysis Program "Global"
The following text describes in detail a typical iteration of the general-

purpose discrete/distributed global analysis program as currently implemented
at the LFD. This process has been divided into 12 logical steps, which will
now be described. (Steps 1 through 3 need to be performed prior to the
beginning of the analysis.)
1. Physical Model Describing the Data Is Established

A wide variety of physical models appropriate for fluorescence spec-
troscopic data have been coded into individual subroutines. These individual
subroutines are accessed through the use of specific “keywords” which the
program “understands.” Some of these routines are very general purpose, such
as the compartmental equation solver (Eq. 5.8), and others are very specific to
one type of model (e.g., anisotropy analysis). A list of the current set
of keywords which the program understands is given in Table 5.1. This table
continues to grow, and the program evolves to the extent that it is able to
interpret (in a rigorous mathematical sense) most of the theory and concepts
that fluorescence spectroscopists employ.
An in-depth description of how a model is specified will now be given.

For convenience, the following description will emphasize the complete
specification of models for the compartmental systems theory aspect of the
global analysis program. An alternative tool for the calculation of fluorescence
observables will be described in Section 5.5.2.
2. The Linkage Patterns Appropriate for This Combination of Experiments

Are Established
The total number of fitting parameters used over an entire data surface
are stored in a vector P of length n. An important consideration when
performing a global type of analysis is that one must provide some type of
supporting logic to tell the fitting program which elements of the fitting vector
are appropriate for a particular experiment. What is often implemented in
single-experiment analysis programs is some type of standard “ordering” of
the parameters within the fitting vector so that the program knows what type
of fitting parameter is in each element of the vector. For instance, when fitting
discrete exponentials to a single experiment, the parameters could be entered
into the fitting vector as
It must be emphasized that when a global multiexperiment analysis is per-

formed, this type of ordering of the parameter vector is usually inedequate.
In a global analysis one needs the following additional set of supporting logic:
(a) Logic must be supplied to tell the program which elements of

the total surface fitting vector are being used for this particular
experiment.
(b) Logic must be supplied to tell the program what type of fitting
function is appropriate for the fitting parameters.
The logic required to inform the program which elements of the fitting vector
are appropriate for a particular experiment can be presented using a matrix
of integer values. This matrix ( M ) (mapping matrix) is dimensioned with its
first index equal to the maximum number of experiments which can be
simultaneously analyzed and its second index equal to the maximum number
of fitting parameters which can be local to a single experiment, and a third
index (which is either 1 or 2) describes whether this number points to a
parameter-vector element or a parameter-descriptor element (to be described
shortly). The third index will be set to 1 when pointing to the fitting
parameter array element and 2 when pointing to a parameter descriptor.
When performing a loop over the entire data surface, the current experiment
index counter (nc) is used as the row index of M [M(nc,·,·)]. The numbers
which are then placed along a particular row of M will then be the indices
of the total parameter fitting vector P which are appropriate for the ncth
experiment. For instance, if the ncth experiment utilizes elements 5, 6, 8, and
10 of the main fitting parameter vector, then M(nc, 1, 1) = 5, M(nc, 2, 1) = 6,
M(nc, 3, 1) = 8, M(nc, 4, 1) = 10. Utilizing the logic in the M matrix, there-
fore, allows one to pass to a subroutine only those vales of the fitting
parameter vector which are appropriate for the jth experiment. The local
fitting terms [LT(i)] for the jth experiment can be extracted from the total
surface fitting parameter vector (P) by using the logic:
In this way the first level of support logic [item (a) above] has been
accomplished in a completely general way. However, one additional logical
step remains. Now that the elements of the total fitting vector appropriate for
any single experiment within the data surface have been obtained, one must
determine what type of fitting parameter this element represents (e.g., is the
fitting parameter a simple rate or perhaps a length distribution parameter or
an activation energy, etc.). Again, there are many types of logic which can be
implemented to perform this function, but we have found the following
methodology very flexible.
Again, an indexed vector can perform this function. One can define a
vector dimensioned to the maximum number of fitting parameter “descriptors”
which can be used to define the entire data surface [D(np)]. Each element of
this vector (D) (descriptor vector) is a string of ASCII keywords which
uniquely describes the functional characteristics of this fitting parameter.
Obviously, many different types of descriptor vectors could be used. We
currently utilize the above formalism because it assists both in making
the source code of the program easy to read (and hence to modify) and
establishes a “natural-language interface” for describing the fitting functions
desired. For instance, a typical keyword descriptor for a simple transfer rate
between compartment 1 and 2 would appear as D(1) = “rate 21.” If that rate
were distributed as a Gaussian, then D(2) = “rate 21 distributed Gaussian.”
If the transfer between compartments involved an energy transfer process,
then D(3) = “length distributed Gaussian 21.” These descriptor terms would
then be “decoded” by the parser subroutine to map to the following fitting
functions:
This type of methodology has proven very useful and flexible and allows
the establishment of a “vocabulary” of keywords which can continually
expand and grow. Eventually, the program evolves to the extent that it
becomes essentially an “expert system” for the analysis of fluorescence decay
models. One of the advantages for an indexed list of fitting functions is that
one does not need to expend a large amount of programming effort to incor-
porate new models into the analysis. In addition, since all of the old model
“keywords” are maintained, a very general purpose nonlinear analysis fitting
environment develops, whereby a wide variety of different models and linkage
patterns can be applied to a particular data surface. There is no longer any
need to stop and redevelop the analysis programs each time one desires to
analyze the data using a new model.
3. Particular Forms for the Functions Linking the Compartments Are Chosen
The appropriate form for the function linking the compartments together
is usually decided by choosing that functional form in which the physical
quantity of interest appears explicitly (eliminate multistep analysis). For
instance, if a multiple-temperature study is being performed, instead of a
rate-space type of analysis, one may wish to globally analyze the data directly
in terms of activation energies and frequency factors. In an energy space,
one useful functional form to describe the excited state interconversion from
compartment 1 to 2 would be
If the activation energy linking the two compartments was distributed, one
would have
If one were performing a multiple quenching experiment (allowing compart-

ment 2 to be the quenched fluorophore and compartment 1 the unquenched
fluorophore), one could define
If the quenching constant was not defined in terms of a discrete quenching

rate, but rather by a distribution of quenching rates, then one could allow
By establishing a wide variety of functional forms for the extremely

complicated ground state and excited state reactions can be examined.
4. The Connectivity of the Compartmental Model Is Established

The connectivity of a photophysical system simply means those sets of
fluorescent molecules which are allowed to interact during the lifetime of the
excited state. For instance, complex decay of fluorescence from systems with
no connectivity simply represents ground state heterogeneity. Mathematically,
a lack of connectivity requires that the T matrix (Eq. 5.8) is completely
diagonal. For excited state reactions, however, there is connectivity between
the various species, and hence some off-diagonal elements of T are nonzero.
Deciding which elements are nonzero establishes the pattern of connectivity
of a system. If some connections between compartments are known to be
absent, the corresponding elements of the matrix T can be fixed at zero. If the
connectivity of the compartments is not known, then various experimental
connectivities may be applied and their corresponding values examined.
Consider the case of a three-state excited state reaction. Three particular
connectivity diagrams are shown in Figure 5.6. These diagrams would
correspond to patterns of zeros in the transfer matrix (Eq. 5.8) as follows:
where correspond to Figs. 5.6a, b, and c, respectively, and x

is some nonzero value. Note how simple it is to examine a wide variety of
different excited state reaction patterns, simply by specifying the connectivity
between the various compartments. One does not need to derive the impulse
responses of these various models (analytically); the eigenvector–eigenvalue
solver simply calculates them numerically for both discrete and distributed
transfer rates.
5. Initiate Global Analysis over the Data Surface

A typical way of assembling the data structure is to treat any high-
dimensional data surface as a series of two-dimensional slices of data. For
instance, within the analysis program there are characteristic data matrices
of the form data(x, nc), where nc represents the experimental curve number
index and x follows the number of “channels” of data collected. Data(5, 10)
would therefore represent the fifth data point in the tenth experiment. One
can now set up an index variable (nc) to keep track of the current experiment
which is being examined. This index will therefore be incremented from one
to the total number of experiments which are being combined in the analysis.
6. Perform Parameter Linking and Function Mapping

Using the current experiment index (nc), the logic described in step 3 of
this discussion is now employed.
The relevant ncth-experiment fitting parameters are extracted from the
total fitting parameter vector and passed to a subroutine using mapping
matrix In addition, the field descriptor specifications of these
parameters are also passed using A typical implementation of the
FORTRAN -77 to perform these operations is as follows:
do 100 i=l, nlocterms

ipointp=m(nc, i, 1)
ipointd=m(nc, i, 2)
LT(i)=p(ipointp)
LD(i)=D(ipointd)
100 continue
c
c ipointp is a pointer into the fitting parameter
c vector ‘ ‘p’ ’
c ipointd is a pointer into the descriptor vector ‘ ‘D’ ’
c p=total data surface fitting parameter vector

c D=character array vector containing the ‘ ‘keyword’ ’
c descriptors for each particular fitting parameter
c LT() is a vector containing those fitting parameters
c appropriate for the ncth experiment
c (Lt: local terms)
c LD() is a vector containing the descriptors associated
c with each particular local fitting parameter
c (LD: local descriptor)
c
c one can now call a ‘ ‘parser’ ’ which decodes the parameter
c values and their associated descriptors to assemble the
c appropriate set of differential equations to solve.
c Given LT and LD, parse returns a fully assembled set of
c systems theory differential equations in the matrix T and
c vector b.
c
call parse (nlocterms, LT, LD, T, b)
c
c
c ...
The matrix T corresponds to the T in Eq. (5.8), while “b” corresponds to the
element in the same equation.
Within the subroutine “parse,” the field descriptor parameters are used to
“decode” the local experiment fitting parameters into proper state space form.
This process involves using the keyword descriptors to locate the appropriate
function which will map the current parameter values to a rate space. The
position identifier is now utilized to find which matrix element of the state
space equation this parameter value belongs.
Once all local experiment fitting parameters have been assembled into the
state space equations, this subroutine returns to the main program. All
elements of the state space equation for the ncth experiment are now fully
defined.
7. Solve the Eigenvalue/Eigenvector Problem and Compute Probabilities†

The matrices T and vector appropriate for this experiment are now sent
to a nonsymmetric eigenvector–eigenvalue solver (e.g., EIGRF , (38) Eispack,(39)
†
Some photophysical problems cannot be solved using an eigenvector/eigenvalue approach and
must be solved with some specialized routines (e.g., transient diffusion effects for energy transfer
measurements such as in Section 5.5.2). In these cases, the eigenvector/eigenvalue routines are
bypassed, and control is passed to a specialized equation solver appropriate for that particular
problem.
or equivalent routine). The inverse negative eigenvalues of the matrix T now

become the observed fluorescence lifetimes for this particular realization of the
fitting vector. For instance, for a two-state excited state reaction one can
denote the current spectral output gain vector at a particular emission wave-
length as SAS1 and SAS2. The eigenvectors associated with two eigenvalues
of this system can be denoted EIG(1, 1), EIG(2, 1) and EIG(1, 2), EIG(2, 2),
respectively. The final amplitudes associated with the two relaxation times
would therefore be [cf. Eqs. (5.6) and (5.7)]:
8. Perform the Integration over the Distributed Parameter (s)

If some parameters are distributed, one additional step needs to be per-
formed before the final amplitude terms associated with the exponentials are
complete. For distributed systems, steps 4–6 will only represent one possible
realization of the distributed system. A “realization” simply means one par-
ticular response of the system out of the total set of the possible responses of
the system (discrete systems have only one realization). Associated with each
realization is a particular probability (e.g., for a Gaussian distributed variable
the realization of a value one standard devation from the means is assigned
a probability of 0.242, while for a uniform distribution all realizations are
weighted by unity, etc.). Therefore, the final amplitude terms from step 6 will
need to be further scaled by the probability of the realization. If a particular
parameter that was Gaussian distributed was currently being sampled one
standard deviation from its mean, then the final amplitudes from step 6 would
need to be scaled as In the case where more than one param-
eter is distributed, the probability associated with a particular realization
depends upon the correlation between the individual probabilities. If the
different fitting distributions are independent of one another, then the final
realization probability is just the product of the individual probabilities.
However, if there is some type of correlation between the probabilities, then
the final realization probability is calculated from the multidimensional
probability function.
These final amplitude terms and their associated lifetimes are then used
to calculate the observed fluorescence using Eq. (5.10) (time domain) or (5.11)
(frequency domain). Integration routines now iterate steps 5 and 6 until some
convergence criterion is met (e.g., the calculated phase and modulation values
or intensity at a particular channel fall by a factor of 5–10 less than the noise
in the actual data). The actual integration procedure occurs in the fitting
space, but the convergence criterion is maintained in the data space. For
instance, when integrating over a distance distribution, or an activation

energy, etc., there is not a simple map between the integration space and the
data space. Therefore, one integrates in energy/distance space, but examines
for convergence criteria in the data space. The limits over which the distribu-
tion function is integrated are user defined. In practice, one usually limits the
integration procedure to approximately 99 % of the area under the distribu-
tion. Discrete summing of many exponential terms to approximate the
integral can be highly inaccurate, especially for short-lifetime distributions.
This type of summing does not produce a truly continuous distribution of
lifetimes. Certain integrals can be calculated analytically, and a new functional
form may be used to analyze the data.
One should note that the fitting space in which the distribution function
is defined is important. For instance, the integration of a uniformly distributed
lifetime from to will yield different results than the integration of a
uniformly distributed rate from When performing analyses in
terms of some distributed parameter, it is very important to consider what
space the distribution originates from. For instance, consider the decay from
a single fluorophore in solution. The lifetime can be written as
If is found to be distributed, the origin of this distribution could reside

in a distributed radiative or nonradiative rate. The rate distribution itself
could result from either a distribution of Arrhenius frequency factors or a
distribution of activation energies, or even both. Any particular distribution
function (e.g., uniform, Gaussian, etc.) applied in these different distribution
“spaces” will each yield a very different set of distributed lifetimes. The choice
of where to place the distribution function is very important and should be
guided both by the types of experiments which are being combined and the
physics of the system being examined. Being constrained to only fitting
distributed “empirical” lifetimes is a severe limitation and should be avoided.
In particular, single-curve analysis via empirical distribution functions is
subject to errors from parameter correlations. This behavior is just like that
seen in single-curve exponentials analyses, and overdetermination is usually
required to validate mean and width estimates.(42, 26)
9. Calculate Derivatives and Set Up the Least-Squares Equations

The result of step 7 then becomes the calculated values for this particular
experiment and the chi-square and residual vector can be formed. The basic
equations required to perform a nonlinear analysis are now assembled (see
Section 5.3.4).
The key distinguishing feature of a “global” analysis is that the nonlinear

data analysis is simultaneously performed over the multiple experiments [see
Eq. (5.16)]. To perform this type of analysis, one must supply an additional
amount of logic which will allow one to combine the elements of Eq. (5.18)
(C and B) when using multiple fitting functions defined over multiple
experiments. There is no unique (or best) way to implement this logic.
The elements of Eq. (5.18) are then formed using numerical or analytical
derivatives (whichever are available for a particular model type). In addition,
a chain-rule set of derivatives can be easily calculated for certain models.
A combination of all three approaches is performed in the current analysis
program. For numerical derivatives, either forward difference or central
difference equations are used (and are user selectable). The least-squares
equations are then assembled. Note that the derivatives calculated from
different experiments pertaining to linked parameters must be summed
together. This can be performed quite easily utilizing the mapping matrix M
in the following fashion (explained using FORTRAN-77 code):
c
c Section of LFD global analysis FORTRAN 77 code which
c assembles the normal least-squares equations using the
c mapping matrix to index the various elements.
c
c DEFINITIONS OF TERMS
c
c nlocterms=the number of fitting parameters for the ncth
c experiment
c derphase (i, j)=the derivative of the calculated ith
c phase data with respect to the jth local
c fitting parameter
c dermod (i, j)=the derivative of the calculated ith
c demodulation data with respect to the jth
c local fitting parameter
c residphase (i)=the ith residual in the phase of the ncth
c experiment
c residmod (i)=the ith residual in the modulation of the
c ncth experiment
c errorphase (i, nc)=the variance of the experimental
c measurement of the ith phase in the
c ncth experiment
c errormod (i, nc)=the variance of the experimental
c measurement of the ith modulation in the
c ncth experiment
c varphase=the variance of the ith phase data point
c varmod=the variance of the ith modulation data point

c ndata(nc)=the number of data points taken in the ncth
c experiment
c ipointl, ipoint2=perform the ''reverse-mapping'' so that
c the nonlinear least-squares equations
c can be assembled
c
do 200 j=l, nlocterms
ipointl=mpoint(nc, j, 1)
do 110 i=l, ndata(nc)
varphase=errorphase(i , nc)
varmod=errormod(i , nc)
beta (ipointl )=beta( ipointl )+
1 residphase(i)*derphase(i, j)/varphase+
2 residmod(i) * dermod(i, j)/varmod
110 continue
do 150 k=l, j
ipoint2=mpoint(nc, k, 1)
do 120 i=l, ndata(nc)
C(ipointl, ipoint2)=C(ipointl, ipoint2)+
1 derphase(i, j) * derphase(i, k)/varphase+
2 dermod(i, j) * dermod(i, k)/varmod
120 continue
C(ipoint2, ipointl)=C(ipointl, ipoint2)
150 continue
200 continue
The matrices C and beta (above) correspond to C and B from the normal
least-squares equations (Eq. 5.18).
10. Finish Loop over All Experiments

Test to see whether the entire surface has been examined. If there are
more experiments in this data set, then increment the current experimental
counter (nc) and go to step 5. Otherwise, proceed to the next step.
11. Get Proposed Parameter Increment for Global Fitting Vector

Solve system of equations for proposed delta parameter increment. Test
whether this new set of parameters decreased the value. If so, decrease
(Eq. 5.19) and proceed until some convergence criterion is met. If increases,
make larger and solve the modified set of normal equations until
decreases.
As an aside, one should note that Eq. (5.18) is seldom solved by matrix
inversion. More efficient equation solvers are available for such symmetric
systems. We have found that the use of the Cholesky decomposition or
“square-root” method (52) is sufficient for almost all data sets. The most robust
numerical method of performing nonlinear least squares is by by passing the
formation of Eq. (5.18) altogether and using the singular-value decomposition
technique (see, for example, Refs. 53 and 54). However, this technique
requires the formation of a matrix of dimension as compared to
matrices with dimension in Eq. (5.18) (N represents the total number
of data points used in the analysis, and m the total number of fitting
parameters). For many global analyses, the data densities are simply too great
(often as many as a million total data points) for the use of the singular-value
decomposition technique for solving the nonlinear least-squares problem.
However, in cases where the analysis has been performed using both techni-
ques (when applied to typical fluorescence decay data/models), identical
results are usually obtained (J. M. Beechem, unpublished results). It should be
emphasized that it is almost always not the mechanics of the minimization
that causes problems in the data analysis, but rather the limited “information
content” of the experiments.
5.5. Case Studies of the Application of Global Analysis to

Experimental Data
5.5.1. Case Study of a Two-State Excited State Reaction
Originally, global analysis procedures emphasized systems having ground

state heterogeneity.(19,20) In these cases, the observed lifetimes represent the
decay rates for each species in solution. The amplitudes associated with each
particular lifetime are also directly related to the excitation/emission spectra
of each emitting state. For excited state reactions, neither of these simple
relationships exists. Global analysts of excited state reacting systems in terms
of lifetimes and their associated amplitudes, therefore, does not directly recover
the “target” parameters of real interest: rate constants for interconversion and
species-associated spectra (SAS). Various levels of empirical and target global
analysis can be performed on these systems and will be described below.
Consider the very simple case of a two-state excited state proton transfer
reaction. Within this model framework, the following two types of overdeter-
mination axes can be easily exploited: multiple emission wavelengths and
multiple pHs. For a global analysis of the data surface,
one should examine which fitting space is most “natural” for this particular
set of experiments. With a global analysis in mind, this question becomes,
which fitting space will minimize the number of fitting parameters neccessary
to describe the fluorescence surface and provide numerical values (and
associated error bounds) of the primary parameters of interest?
Consider the global analysis of two pH experiments with four different
emission wavelengths. This data surface can be represented in terms of
impulse response parameters as:
From the completely empirical model of a sum of two unrelated

exponentials, this total decay surface can be described using total
fitting parameters. Although each data set would be examined in terms of a
four-parameter minimization, the net result after the analysis of the eight
experiments would be the same as if a 32-parameter “nonlinked” minimization
was being performed.
From the structure of a two-state excited state reaction, it is known that
the fluorescence lifetimes are invariant as a function of the emission
wavelength. Therefore, one can perform a global analysis of each particular
pH experiment by linking the observed lifetimes as a function of emission
wavelength. This reduces the dimensionality of the fitting space from 32 to 20
(two sets of two lifetimes plus two sets of four values). The lifetimes and
amplitudes between different pH experiments cannot be linked because they
are a function of the kinetic system as a whole and will change with pH.
If the fitting space is removed completely from space, a further
reduction in the number of parameters results. Using the systems theory
equations (Eq. 5.8), a fitting space can be defined which consists of the rate
constants of interconversion the lifetime of each
emitting state and the spectra associated with each
emitting state For this type of analysis, knowledge of
the boundary conditions may be required.(55) The dimensionality of the
fitting space is now reduced to just 12 fitting parameters. Simply by imposing
the physical model “two-state excited state proton transfer,” the fitting space
has been reduced from 32 to 12 dimensions. This radical reduction in fitting
space can result in a transformation of the error surface from a rather ill-
defined flat surface to a very well defined, nearly quadratic error surface.
This type of analysis was applied to the excited state proton transfer of
-naphthol. (24) Time-resolved data were collected at pH 2.15 and 3.0 at 75
emission wavelengths in each case (this number of emission wavelengths is
only necessary if high-resolution species-associated spectra are desired). A
global analysis was now performed by simultaneous analysis of a data surface
consisting of fluorescence versus time versus emission wavelength versus pH.
The total number of fitting parameters are the four rate constants plus two
species-associated spectra (a total of 154 fitting parameters!). It should be
noted that the rate constants can be completely determined using only two
emission wavelengths, so that only six fitting parameters are needed for that
analysis. However, it was desired to attempt to recover the high-resolution
SAS for this system so that all of the emission wavelengths were utilized. The
rate constants recovered from this system are shown in Table 5.2.
One can see that the simultaneous analysis of only two pH values was
sufficient to resolve the rate constants of this system, compared to the
extensive pH study performed previously.(56) The global analysis results, of
course, only strictly pertain to the pH region in which the study was actually
performed.
One reason the 2-naphthol case was reexamined was because the species-
associated spectra can be experimentally determined, and therefore the SAS
recovered from the analysis (using 154 fitting parameters) can be directly
compared with the known spectra. The results of this comparison are shown
in Figure 5.7. The spectrum associated with each emitting state recovered
from the analysis program is plotted along with the experimentally determined
SAS (pH = 0 yields only naphthol, pH = 13 yields only naphtholate). From
these results, it appears that not only all the rate constants for the reaction
can be recovered, but also each individual SAS. Therefore, all 154 fitting
parameters are uniquely recoverable and can be shown to faithfully represent
physical quantities.
For many biochemical systems of interest, it will not be possible to alter
conditions so as to obtain the individual SAS experimentally in a direct way

(i.e., extreme pH regions will destroy sample). Application of a “target” global
analysis as described above can decompose these complicated spectra into
their time-invariant components. Davenport et al.(57) have demonstrated a
DAS remixing scheme suitable for some of these cases. An alternative multi-
step approach to obtain SAS has recently been described by Löfroth. (45)
Consider a hypothetical extension of the above two-state excited state
reaction system, where the proton-transferring probe is now covalently labeled
to a protein or biological membrane. It may be found that the observed
lifetimes of the fluorescent probe as a function of pH and emission wavelength
are more complicated and are better fit using a distribution of lifetimes rather
than discrete lifetime values. A whole series of empirical analyses can now be
performed using distribution functions to describe the lifetime components.
The end result of these analyses, using either parameterized or maximum-
entropy methods, would be a series of distributions as a function of pH
and emission wavelength. However, the primary quantities of interest, rate
constantst and SAS, would not be determined by this type of empirical
analysis.
One may feel that an empirical analysis is all that can be performed on
a system as complicated as this. However, various physical models can still be
applied using the systems theory and global analysis framework. The concept
of a discrete pH value inside a protein may not be particularly valid, and,
during the lifetime of the excited state, the probe may indeed experience a
distribution of proton concentrations (activities). With this model in mind, a
global analysis of the multiple pH and emission wavelengths can be performed
by altering the function will
represent the distribution of protons within the interior of the protein and
will be an actual fitting parameter. Within this model framework, one can
maintain a structure which is consistent with the probe’s solution behavior,
yet allows an additional level of complexity due to its now more complicated
(protein/membrane) environment. A similar reduction in parameter space
compared to empirical fitting is still obtained; however, since is now
distributed, both lifetimes and their associated amplitudes will also appear
distributed due to the nature of the eigenvector eigenvalue solution of
Eq. (5.8). Negative-amplitude terms will appear “naturally” due to the fact
that the excited state reaction distribution structure is being imposed at the
level of the differential equations, not their solutions. The final results at
the end of the analysis would contain, in addition to all the discrete fitting
parameters, a distribution parameter which would reflect the actual proton
distribution in the interior of the protein.†
†
Since pH is a macroscopic parameter (by definition), the use of proton concentration should
perhaps be replaced with that of site-specific reactivities.
This type of analysis result clearly represents the philosophy behind the
global analysis step: transform a series of empirical fittings into an
internally consistent physical result. The unlinked empirical analyses
represent the best model-independent representation of a given set of experi-
ments. However, the solutions, although mathematically correct, may have
no physical analogue. The global analysis procedures do not compete with
the empirical single-experiment analysis approaches; they simply represent
the logical next step in a multistep process which transforms data into
“biochemical information.”
5.5.2. Distributions of Distances and Energy Transfer Analysis
The advantages of the use of global/target analysis can also be illustrated

for the interpretation of resonance energy transfer experiments. Resonance
energy transfer is well established as an experimental method for estimating
distances or distance changes in macromolecules.(58–62) The time dependence
of the rates of nonradiative energy transfer between probes attached to unique
sites on a biopolymer is a function of both the distribution of the distances
and the rates of interconversion between the distances. One can model this
process, using the following equation(63)
where is the equilibrium distribution of the energy transfer pair,

is the interprobe diffusion constant, r is the
interprobe distance, is the lifetime of donor, and is the associated
amplitude.
The first term on the right-hand side of Eq. (5.31) represents both the
spontaneous decay of the donor and the decrease of excited donor concen-
tration due to nonradiative energy transfer. The second term represents the
replenishment of the depleted fractions by the Brownian motion of the labeled
segments. The distance distribution, obtained by solving Eq. (5.31)
represents the time-dependent reduction of the excited donor population
[normalized to ] at each distance fraction, taking into account both
distance distributions and interconversions. In spite of the fact that the time
dependence of the rate of energy transfer is clearly a function of the donor–
acceptor distance distribution and fluctuations between the distances that occur
during the lifetime of the excited state, attempts to recover both the distance
distributions and the diffusion rates have proved to be elusive. A method has
recently been developed by Beechem and Haas,(64) which performs a global
analysis of donor and acceptor decay in terms of both distance distributions
and diffusion rates as the target fitting parameters. This method has overcome
many problems associated with close correlation between fitting parameters.

Both distance distributions and intramolecular diffusion parameters were
recovered with high statistical significance.
Initial time-resolved fluorescence experiments aimed at obtaining the
spatial distribution and the diffusion rate information from the available data
employed a multistep analysis process similar to that used for analysis of
other excited state reactions. (58–60) The decay time of the donor was obtained
in the absence of acceptor to determine the donor lifetime and amplitude. This
decay time then became a quantity that was fixed in the next stage of the
analysis involving the decay of the donor in the presence of the acceptor.
Analysis of experiments at high viscosity was used to obtain distance distribu-
tions in the absence of diffusion. This recovered distance distribution was then
fixed in a subsequent analysis of data obtained at low viscosity to yield the
diffusion coefficient. This type of multistep analysis leads to difficulties in error
propagation and involves restrictive assumptions involving extrapolation of
temperature- and viscosity-dependent data.
Upon treating these data with a global analysis algorithm, the very ill
defined error surfaces from “donor-only” analysis are replaced by well-defined
single minima (see Figure 5.8). The radical transformation of the error surface
can be rationalized as follows. The interprobe distance distribution of an
ensemble of molecules with an excited donor probe at the time of excitation
will be identical to the equilibrium distance distribution. A very rapid decay
of the donor excited states occurs for the fraction of molecules with short
interprobe distances because of the steep dependence of transfer
probabilities. Thus, the donor emission of the fractions of molecules with the
shortest interprobe distances can decay very fast. It is difficult to obtain an
exact quantitation of the amplitude contributions of these very fast decay
components (they disappear at quite early times). These parameters are thus
recovered with poor accuracy from analysis of the donor fluorescence decay
curve alone. The acceptor emission, however, recovers these parameters with
high sensitivity, due to convolution of the transferred photons with the
acceptor impulse response, which effectively “spreads” this information out
over a much larger time interval. A similar argument holds for the weighting
of the emission from the fractions with large (relative to ) interprobe dis-
tances. These long distances contribute a weak signal to the acceptor emission
but are well weighted in the donor emission. Thus, the simultaneous use of
both energy transfer donor and acceptor decay data leads to a significant
improvement in the ability to recover the desired fitting parameters. A further
enhancement of parameter recovery can be achieved by combining experi-
ments done at different temperatures and/or viscosities or done in the presence
of different concentrations of quenchers. The combination of these various
experiments into a single data analysis provides an ideal example of the
“global philosophy.”
5.6. Anisotropy Decay Data Analysis
5.6.1. General Equations and Experimental Linkages
The emission anisotropy r(t) is defined as
where and are the time-dependent emission components measured

parallel and perpendicular to the polarization of the excitation light, respec-
tively. From the definition of the emission anisotropy and the fact that the
total fluorescence for a macroscopically isotropic sample is given by
the individual polarized components can be expressed as:
These expressions reveal that both and are determined by the same
set of fitting functions [i.e., f(t) and r(t)] and hence the same set of fitting
parameters. This statement can be made for any polarized intensity obtained
under any combination of polarization directions in the excitation and the
emission. The intensity corresponding to excitation and emission polarizers at
angles and with respect to the normal to the excitation–emission plane
is given by
The time dependence of the anisotropy is expressed by the ensemble average

of the angle between the absorption dipole at time zero and the emission
dipoles at time
where is the second-rank Legendre polynomial, and the angle brackets

denote the ensemble average; and are unit vectors along the transition
moment of the absorption at time t = 0 and of the emission at time t, respec-
tively.
From the general expression for the decay of the emission anisotropy
(Eq. 5.37), various specific cases can be calculated. The anisotropy decay
of a general rigid asymmetric body in an isotropic environment is described
by a sum of five exponentials.(66–68) In many cases, these five exponentials can
be approximated by a sum of three exponentials.(69) When approximating
the hydrodynamic shape of a fluorescent system in terms of an ellipsoid of
revolution, one has(28):
where
where and are the angles made by the absorption and emission dipoles
with respect to the symmetry axis and where is the angle between their
projections in the plane perpendicular to the symmetry axis. The corre-
sponding rotational correlation times are given by:
where is the rate of rotation about an axis perpendicular to the symmetry

axis, and is the rate of rotation about the symmetry axis. Note that there
are three rotational rates but only two physical parameters: and
The anisotropy decay of a body subjected to diffusion in a hindering
potential of arbitrary form cannot be expressed in closed form (as described
above). In these cases, the anisotropy decay is found to be described as an
infinite number of exponentially decaying functions. An empirical expression
consisting of a limited sum of exponentials and a constant term has often been
used to approximate this decay:
By examining trends in the parameters and along various experimental

axes, various conclusions can be made concerning the physical parameters
which describe the system (e.g., critical temperatures, diffusion coefficients,
etc.). The problems inherent in this type of two-step analysis are identical to
those already discussed for total intensity decay data.
A sequential method of analyzing polarized fluorescence decay data
involves the formation of two related decay curves, S(t) (the “sum” curve)
and D ( t ) (the “difference” curve):
with
where l(t) is the measured excitation function, and denotes the convolution
product.
The sum curve is proportional to the decay of the total intensity and
contains no fitting parameters for the anisotropy. In the sequential analysis,
the total fluorescence decay parameters are determined first from nonlinear
minimization of S(t). The results from the S(t) analysis are used as fixed
parameters in the subsequent analysis of the D(t) curve.
As an alternative to this “sum-and-difference” analysis, and can be
simultaneously analyzed for the parameters of S(t) and r(t) using complete
linking of parameters. (70,71,23,25) This method has certain advantages with
respect to the “sums-and-difference” analysis approach and has been adopted
for all of the current global analysis programs. What will be discussed in
the following sections will be the global analysis of multiple anisotropy
experiments, keeping in mind that each individual experiment requires
simultaneous analysis of a single and measured decay.
The actual fitting programs for both empirical and model-dependent
global analysis can be implemented using the general methodologies described
in detail in Section 5.2. One merely defines an additional set of anisotropy-
specific “keywords.” A description of global anisotropy analysis and essential
program code are also given elsewhere.(23)
In the following sections, we present a series of examples to illustrate how
global analysis has been used to study some complex rotational behavior.
Some of these cases are of general applicability, whereas other cases are
specific to the study of particular biological systems.
5.6.2. Changes in Anisotropy Data Collection Schemes
Since the development of the global analysis software, we have found

that certain problems associated with the actual hardware collection of
fluorescence data have been eliminated. In the case of anisotropy data, the
analysis of data obtained using the simultaneous collection of and
(“T-format” time-resolved anisotropy data collection) proved difficult because
of the requirement that the timing characteristics of both phototubes be
identical. However, when performing a global analysis on and
each with its own excitation profile and timing calibration, there is no need to
match the two phototubes.(23, 74) This matching is both difficult to accomplish
and unnecessary.
In global total intensity decay analysis, experimental use of multiple
timing calibrations has proved useful in determining multiple, widely varying
decay rates.(72) A similar approach can also be used in anisotropy analysis. To
resolve multiple rotations occurring on widely differing time scales, multiple
anisotropy experiments are performed under differing timing calibrations.
These multiple timing calibration experiments can be combined into a single
global analysis in which one finds an internally consistent set of rotational
correlation times. In this way, one is not forced to fix various rotational
correlation times when performing a sequential analysis over differing time
domains. Also, instead of using very large single data sets, obtained over
many channels using a timing calibration which represents a compromise to
resolve both fast and slow motions, it may be much more convenient to
obtain a series of small sets collected at multiple timing calibrations.
The above are simply two examples in which the data analysis metho-
dology has directly influenced the data collection aspects of anisotropy
experiments. In both cases, rather difficult “hardware”-related problems have
been solved using a “software” approach.
5.6.3. Associative versus Nonassociative Modeling of Anisotropy
The problem of associative versus nonassociative decay is related to a

molecular description of the species present in the experiment. This problem
arises when a multiexponential decay of the fluorescence intensity is combined
with a multiexponential decay of the fluorescence anisotropy. In the non-
associative case, the set of correlation times in the anisotropy decay is
common to each of the relaxation times in the fluorescence decay [i.e., all
cross-terms in the formation are nonzero]. In the associative
case, each particular lifetime may be “associated” with a specific rotational
correlation time.
Both types of associations can be expressed mathematically by the

following expressions (1,74) :
The matrix L is defined as
It is very difficult to discriminate associative from nonassociative decay

based on a single anisotropy experiment, as equally good fits result in many
cases. The model-testing capability of global analysis, however, can be helpful
in the discrimination of these two cases. Associative and nonassociative
models behave very differently when one performs a global analysis of the
polarization decays collected at multiple emission wavelengths. For instance,
when the species of the polarization decays collected at multiple emission
wavelengths. For instance, when the species in an associative case have
different emission spectra, one can perform a global analysis by linking
(which may be independent of emission wavelength for a given model, e.g.,
ground state heterogeneity) and only allowing the to vary. Using this
linkage, proved to be very sensitive to discriminating (in a statistically
justifiable manner) associative versus nonassociative behavior. (23,74,72)
5.6.4. Anisotropy Decay-Associated Spectra (ADAS)
Even in the absence of heterogeneity in total intensity decay, steady-state

spectra can still be resolved into underlying components that the various
species have different anisotropy decays.(75) Consider a fluorescent probe
which partitions into a lipid bilayer having two distinct phases (a “fluid”
phase and a “gel” phase). The emission spectra of the dye in the two phases
may be distinct, yet the fluorescent lifetimes may be very similar. One would
like to be able to decompose the emission spectrum into the contributions
from the gel and fluid phases.
The different spectra can be obtained from a data surface that is
constructed by recording anisotropy decays across the emission band. This
can be demonstrated as follows. In an associative model, the parallel polarized
intensity component for species is given by
where is the total intensity decay of species k, factorizing

spectral- and time-dependent parts. Similar expressions can be given for other
polarized intensities. The desired spectra can be recovered from
data of this type, even in the case that (i.e., no total-intensity decay
heterogeneity), because the multiple species are associated with different
anisotropy functions.
To resolve these anisotropy decay-associated spectra (ADAS), one may
perform a global analysis of this data surface by linking the fluorescence
relaxation times and associated anisotropy functions across
the emission band, allowing only the spectral components to
vary. A plot of the individual as a function of emission wavelength then
represents the emission spectra associated with a particular anisotropy decay
function and hence the ADAS. ADAS have been successfully obtained for
fluorophores incorporated into rotationally distinct environments.(75– 77)
5.6.5. Multidye Global Anisotropy Decay Analysis
The rotational motion of a protein is often monitored through the use of

extrinsic chromophores covalently attached or adsorbed to the protein.
However, due to the geometry of dye binding to the protein, the measured
anisotropy from the extrinsic probe may preferentially emphasize particular
aspects of the protein motion. For instance, extrinsic dyes whose dipoles align
preferentially along the long axis of the protein tend to yield longer rotational
correlation times since they are primarily sensitive to rotation about the
short axes. The net result is that multiple dyes attached to a single protein
may yield widely different rotational correlation times. An explicit example is
the change in mean rotational correlation times observed with various
fluorophores bound to horse liver alcohol dehydrogenase (HLADH). (25) Since
each particular dye may bind to HLADH with a different direction of the
absorption/emission oscillators with respect to the principal diffusion axes,
each dye will report a mean rotational correlation time which will be a
different weighted average of the correlation times of the protein. In the case
of HLADH, mean values were found to differ greatly as a function of the
particular reporter dye that was bound. A global analysis was then performed
on the multiple dye experiments, in terms of resolving an internally consistent
set of rotational correlation times over the different experiments. The multiple
correlation times obtained from this global analysis were in reasonable
agreement with those predicted from the crystal structure. In this way, the
anisotropy character of the overall motion of a globular protein was uncovered.
5.6.6. Distributed Lifetimes and Distributed Rotational Correlation Times
The polarized intensity of a sample containing several species is just the

sum of the individual polarized intensities. A natural extension to multiple
discrete lifetimes and rotation rates is distributions of these decay parameters.

A general, empirical fitting of fluorescence anisotropy decay data surfaces is
accomplished by distributing the relaxation times in the expression for total
fluorescence and anisotropy. The parallel-polarized intensity component is
then given by
where is the multidimensional probability associated with the

angular dependence, rotational dependence, and lifetime dependence of the
distribution function. For the nonassociative case, one can replace
with
Distributed rates may be expected for fluorophores in anisotropic media
for a variety of reasons; the simplest would obviously be simply having a very
heterogeneous rotational environment. We will also show, however, that when
fluctuations in the environment occur during the lifetime of the excited state,
a distribution of rotational rates will also result. A physical model for fluores-
cent probes in membranes which leads to distributed rotational correlation
times will be presented in Section 5.6.8.
5.6.7. Multiexcitation Anisotropy Experiments
Anisotropic rotations in small molecules were first determined by Weber

et al.(78,79) It is often very difficult to detect anisotropic rotation of a molecule
because the rotational correlation times do not differ by a large extent. As a
result, the analysis of a single anisotropy experiment often yields only the
average of the multiple correlation times. Barkley et al.(28) described the use of
multiple excitation wavelengths, which alter but yield the same to aid
in unraveling complex anisotropic rotations. This useful overdetermination
arises because the are only functions of the relative orientation of the
transition moments in the molecular frame [see Eqs. (5.38)–(5.41)]. If one
does not obtain the same values upon excitation into different absorption
transitions, anisotropic rotations must be considered. The relaxation times of
the anisotropic motion can be obtained accurately by global analysis of the
anisotropy decays at different excitation wavelengths by linking the correla-
tion times. The different preexponential factors at the various excitation
wavelengths result in a different weighting of the correlation times. This
approach has been applied to recover the anisotropic motion of perylene and
9-aminoacridine.(28,23) Perylene is very anisotropic rotator, so the use of
global analysis simply provides a rigorous way of recovering the most inter-
nally consistent ratio of diffusion coefficients for the molecule
In the case of 9-aminoacridine,(28,23) however, it was impossible to determine from
single-experiment analysis whether this molecule was an anisotropic rotator.
However, with global analysis, one could determine, in a statistically signifi-
cant manner, that this molecule was not an isotropic rotator and recover
Recent work (performed in the frequency domain) suggests that
the rotational behavior of perylene may be even more complicated.(80)
5.6.8. Example of Distributed Rotations: Fluorophore Rotations Gated by Packing

Fluctuations in Lipid Bilayers
Davenport et al.(8l,82) investigated the fluorescence anisotropy of the

long-lifetime probe coronene in liposomes. The unusual behavior
observed with this probe led to the suggestion that the emission anisotropy is
sensitive to “packing fluctuations” in lipid bilayers.
Two levels of modeling, one discrete and one distributed, were performed.
For the discrete model the membrane system was treated in terms of a simple
gel–fluid equilibrium, characterized by two rate constants, and
These rate constants represent the rate of lipid exchange from the gel to the
fluid compartment and from the fluid to the gel compartment, respectively.
It is assumed that little rotation of the probe takes place in the gel state and,
also,
Within this framework, probe molecules found initially in gel are
(83)
characterized by an “effective” rotational correlation time
The emission anisotropy is “gated” by the transfer of probe into the fluid
state. Those probes will thus rotate only by going through a “local melting”
process. If all probes participate in this equilibrium, the decay of the emission
anisotropy can be expressed as
where and
When is large, can take the place of found in previous models.
One may extract and terms as a function of temperature. Trends for
the and terms can be analyzed in a sequential manner or by global
analysis. Examination of these intermediate parameters revealed behavior
not consistent with this model framework. Thus, an alternative framework
was developed which predicts a distribution of melting rates. Jahnig (84) has
discussed the use of a Landau model for packing fluctuations. A model based
on rotation “gated” by such packing fluctuations was proposed. In this model,
the free energy potential shapes for dipalmitoyllecithin (85) were used by
Davenport et al.(81) to model the emission anisotropy behavior of coronene in
liposomes as a function of temperature. A target parameter space, consisting
of free energy profiles, a gating factor, and a diffusion coefficient, was estab-
lished for the analysis. Simulation of the observed emission anisotropy of
coronene as a function of temperature was performed. This use of Landau
theory provided a method whereby functional “linkages” could be constructed
in a physically meaningful manner over the temperature axis.
5.7. Error Analysis and the Identifiability Problem
The result of a nonlinear fit to data is a series of numerical values

associated with the fitting parameters of the proposed model, the value of the
reduced chi-square, a surface of residuals, and possibly a surface of auto-
correlations. The recovered numerical values of the fitting parameters are
essentially useless unless some type of error analysis is performed. Historically,
only the errors associated with the recovery of the fitting parameters have
been examined. What we will describe in the following sections is the
examination of two types of error:
1. Are the recovered parameters unique, or can they take on widely
differing values and still yield the same identical response (and hence
chi-square)?
2. What is the range of possible values that these parameters could
assume and still yield chi-square values consistent with that obtained
at the minimum?
The first item is directly related to the identifiability of the model, while the
second addresses error analysis within a model. A description of both the
identifiability problem and the error analysis problem will be discussed in
this section. Although the error discussion will be completely general, the
identifiability study will focus only on nondistributed compartmental models.
5.7.1. The Identifiability Problem
The concept of identifiability (in terms of typical fluorescence decay

experiments) can be stated very simply: Does the series of experiments being
performed contain enough information to provide a unique solution for the
set of global fitting parameters (within the model structure) which is being
applied to the data? Figure 5.9 schematically depicts an identifiable and a
nonidentifiable set of models. In the nonidentifiable model “A,” various sets of
numerical values for the fitting parameters (denoted as specific values

2, 3, …, N) each yield identical impulse response functions. Therefore, one
could never differentiate these sets of parameters from each other, and hence
this model is said to be nonidentifiable. In contrast, all specific sets of numeri-
cal values associated with model “B” yield unique impulse response functions.
Therefore, this model is termed identifiable. Note that no statement is being
made concerning how “closely” the impulse responses resemble one another,
only that they are not identical. From this figure, it is apparent that one does
not need to discuss the concept of identifiability if one only fits each experi-
ment in terms of the parameters of an impulse response function ( and ).
However, with the use of global analysis, problems are often reparameterized
so that the actual fitting parameters are no longer a direct description of the
impulse response function. In these cases, it is important to determine whether
the recovered parameters provide a unique mapping to a set of impulse
response functions.
Consider the following simple two-state excited state reaction with
and allow only
compartment 1 to be populated in the ground state (can be generalized to any
combination of initial ground state populations). The above system is
examined at two different emission wavelengths toward the blue and red edges
of the emission spectrum The impulse
response functions for these two experiments are
The above hypothetical data set can be analyzed in terms of a sum of

unrelated exponentials. Depending on the noise in the data, one should be
able to recover a total of four amplitudes and two lifetime terms. From
examination of the results of this empirical analysis, it should be evident that
a two-state excited state reaction is occurring (note: negative amplitude on
red edge).
In this case, the “target” parameters of interest are probably not the
amplitudes and lifetimes, but rather the rate constants for interconversion,
the decay times, and the spectra associated with the two emitting species.
One therefore reparameterizes the problem, in terms of the “target” fitting
parameters: When performing the global
analysis in terms of these target parameters, it will be found that there is an
infinite set of target parameters which will yield the two impulse response
functions given by Eqs. (5.55) and (5.56). One element of this set is
and
(compare with above synthesized values). Depending on the
type of error analysis performed on these target parameters, one would find
very large error bars associated with these values. These large error bars are
not due to “noise” in the observed data (infinitely accurate data would have
exactly the same error limits). Instead, these errors result from the fact that
there is no unique set of target parameters which map into the impulse
response functions (Eqs. 5.55 and 5.56). However, if one can experimentally
alter either or (by changing pH, concentration, etc.),
then analysis of the combined data surface of fluorescence versus versus

pH (or concentration) versus time/frequency will yield a unique solution to the
problem (assuming knowledge of the ground state absorbances).
5.7.2. Identifiability Study Using Laplace Identifiability Analysis
The above example (Section 5.7.1) is a single example of a model which

is not identifiable. Identifiability analysis attempts to predict what combi-
nation of experiments is required to uniquely determine a particular model
from a given set of data. There are numerous techniques which have been
developed (mostly within the field of systems theory and compartmental
analysis) to determine the identifiability of a particular model. What will be
presented below will be one particular identifiability approach, to allow
readers to obtain a better understanding of how to determine the “information
content” of a given set of fluorescence experiments.
In the case of a linear problem, one can determine the possibility of a
unique solution to a problem, by simply counting the number of unknowns
(to be determined) and the number of independent equations. If the number
of unknowns exceeds the number of independent equations, then no unique
solution is possible. This basic idea can be extended to examine the
identifiability of particular compartmental models of fluorescence decay data
through the use of the Laplace transform. It should be emphasized that the
following analysis is completely independent of the noise on the data.
The basic systems theory equation (which is utilized by the global
analysis program for all discrete/distributed compartmental models) can be
written:
[see Eq. (5.8) for definition of terms]. The observed fluorescence can be
written as
where F is the observed fluorescence, A is an output matrix of spectral

contours, and X is the time course of each fluorescent species in solutions
[vector form of Eq. (5.9)]. Taking the Laplace transforms of Eqs. (5.57) and
(5.58) yields (given a zero initial state):
where s is the Laplace transform parameter. Solving for the Laplace transform
of the concentration of each individual species yields
(given that the inverse exists). The Laplace transform of the fluorescence can
therefore be written:
The factor is termed the transfer function matrix because it

relates the input of the system with the output of the system
The analytical form of the transfer function will be useful in deciding the
number of unknowns which can be determined from a particular set of
experiments.
Consider the very simplified case of a two-state excited state reaction in
which only species 1 is excited, and only species 1 is observed. This would
correspond to and The transfer function is therefore
Expanding out this equation and substituting the explicit rates into yields
The usefulness of Eq. (5.64) is that it predicts what combinations of rate

constants will yield identical transfer function matrices, and therefore identical
impulse response functions (see Figure 5.9). From Equation (5.64), one has
that whenever the following relationships are satisfied, identical impulse
response functions will occur:
Therefore, from our model (two-state excited state reaction, known input
and output vectors) one can derive only three independent relationships (Eqs.
5.65–5.67), but there exist four independent fitting parameters (the four rate
constants). Therefore, one has determined a priori that a global target analysis
of this particular experiment directly in terms of the rate constants of the
system is not possible. This result is completely independent of the accuracy
of the collected data.
One can proceed with this type of approach to examine the identifiability
of the two-state excited state reaction, but in this case consider the combina-
tion of two fluorescence experiments performed by altering one of the rate
constants for interconversion (e.g., changing the pH for excited state proton
transfer). In this case, an additional transfer function is generated, with
altered to where [M] is the concentration of interactant. This
additional transfer function generates a new set of independent equations
(similar to those above). By combining the number of independent equations
from experiments 1 and 2, one can determine that the number of unknowns
in the system is now five. † The number of independent equations generated
from this combination of experiments is also five. From these two experiments,
then, one has a set of five nonlinear equations in five unknowns. Since these
equations are nonlinear, one is not guaranteed that there is a solution;
however, the possibility exists that this system is identifiable. In fact, we know
from the study of the excited state reaction of (see Section 5.5.1)
that this type of system can be uniquely recovered.
More complex models with additional compartments are even less likely
to be identifiable than the two-compartmental model described above, as
they allow for many different connectivities between the compartments. The
number of compartments used in the analysis, of course, cannot exceed the
number of relaxation times which can be resolved by the global analysis.‡
If the dependencies of the rate constants on concentration are unknown, this
will create additional identifiability problems.
The structural identifiability problem is well known in the field of com-
partmental modeling(86,44,87,88) and is still a subject of intensive study. For a
detailed example of the use of identifiability analysis applied to fluorescence
decay data, see the study of Ameloot et al.(27) From this study, no single set
of experimental conditions was found to uniquely determine the system
parameters for all types of two-state excited state reactions. In all cases,
at least two different concentrations of interactants are needed. This is a
necessary (but not a sufficient) condition for identifiability. The set of fluo-
rescence lifetimes alone may, in some cases, resolve all of the rate constants.
In these special cases, neither normalization between experiments nor know-
ledge of the absorption vector is required. In most cases, the information
content of the preexponentials associated with each particular lifetime must be
incorporated. Multi-emission or multi-excitation wavelengths may also be
essential to obtain complete identifiability. Identifiability may also depend on
the particular values of the system parameters.
Fluorescent systems may also be “partially” identifiable. When only a
†
If the effect of [M] on the rate constant is known, then there will be only four unknowns.
‡
Hence, simple linkages over emission wavelength are still useful to identify the dimensionality
of the system.
single-ground-state compartment is excited directly, the SAS of the first

compartment is always uniquely identifiable. This SAS can be obtained by
just summing at each emission wavelength the preexponentials resulting from
the global analysis in terms of linked lifetime. (57) If several compartments are
initially excited, this sum reflects that initial mixture. This result also follows
directly from an identifiability study using the technique described above.
Upon performing that analysis, the SAS of compartment 1 is always uniquely
isolated from the other sets of coupled equations and, hence, can always be
uniquely recovered.
Another example of a partially identifiable case is taken from an
anisotropy study. It can be shown that fluorescence anisotropy data for a
probe in an anisotropic medium can be analyzed in terms of the rotational
diffusion coefficients and the order parameters of second and fourth rank, that
is, and In some cases, there exist two values for at the
same values of the rotational diffusion coefficients and of This
ambiguity can only be resolved by performing the same kind of studies on
oriented samples. Oriented sample measurements excited and measured at
various angles in space may provide an ideal setting for a global target
analysis of complex rotational motion.
Recent work on identifiability by Eisenfeld(90) has shown that by
examination of the determinant of (where J is the Jacobian matrix of the
transformation of the fitting parameters to the observable parameters), one
can determine whether a particular model is identifiable or not. Examination
of the rank (and correlation analysis) of the elements of the matrix “C”
[Eq. (5.19) with can also yield important identifiability information.(91)
This recent work should provide the necessary framework to integrate
general-purpose identifiability tests into the analysis software. Additions of
this type are currently being entered into the global analysis software.
Therefore, when a global analysis in terms of a specific set of target
parameters is performed, the identifiability of this particular parameterization
should be considered. This type of analysis is relatively “new” to the field of
fluorescence spectroscopy, but the insights which this technique can offer make
these types of studies very important. In the case of excited state reactions,
experiments must be performed which can alter the equilibrium between
the various species. This condition, however, does not guarantee a unique
solution. For many experimental configurations, the identifiability problem
will be very difficult to solve analytically, and one must resort to some type
of rigorous error analysis to test for nonuniqueness.
5.7.3. Error Analysts
The values of the parameters recovered by a particular analysis should

always be reported with their corresponding errors. The level of rigor on
which the error analysis is performed depends on only a single factor: the size
of the region of the error surface which is explored.† One can classify the
various error analysis algorithms as follows:
1. Linear approximation: None of the error surface is directly explored.
The information contained in the curvature matrix at the chi-square
minimum is utilized to calculate the uncertainties in the recovered
parameters, assuming no correlation between the various fitting
parameters.
2. Unidimensional search: Directed searches along each parameter axis,
not allowing any of the other fitting parameters to vary.
3. Directed search: Directed searches along the eigenvectors of the
curvature matrix at the minimum.
4. Exhaustive search: Directed searches along each parameter axis,
allowing all other parameters to vary so as to obtain a minimum
chi-square at each point.
Each of these error analysis methods is diagrammatically depicted in
Figure 5.10 (for a two-parameter analysis of “P” and “Q”).
In the linear approximation, the errors on the recovered parameters are
estimated utilizing the square root of the diagonal elements of the inverse of
the curvature matrix see Eq. (5.18)] at the chi-square
minimum. This type of error analysis is strictly valid only for linear models
and assumes that there is no correlation between the individual fitting
parameters. This methodology always produces a symmetric error result
and should always be regarded with a large amount of skepticism. These error
estimates will always predict the smallest amount of uncertainty (see shaded
region in Figures 5.10 and 5.11).
The unidimensional search algorithm simply calculates the observed chi-
square by sequentially altering the recovered fitting parameters along each
independent axis. None of the other fitting parameters are allowed to vary
during this process (no correlations between the parameters are taken into
account). The region of the error surface which is examined by this approach
is schematically represented as the lines A–B and C–D in Figure 5.10.
The error bars recovered from this analysis may be asymmetric. The major
limitation in this error analysis technique is that no correlations between the
fitting parameters are allowed.
In the directed search algorithms, an attempt is made to take into account
some of the correlation between the fitting parameters. By calculating the
†
Of course, knowledge of the chi-square surface over the entire domain of the fitting space would
provide all of the information necessary to establish the error bounds and identifiability.
However, this type of error analysis is usually not possible (too computationally intensive), and
one needs to resort to examination of the chi-square surface along particular axes.
eigenvectors of the curvature matrix at the minimum chi-square, one effectively

“rotates” from the fitting parameter frame to a frame of reference directed
along the regions of greatest covariance between the fitting parameters.
Directed searches in these directions can therefore utilized to examine the
errors in the recovered parameters. These directions are schematically depicted
as the lines E–F and G–H in Figure 5.10. Note that asymmetric error
estimates can be obtained from this analysis, and by rotating to this particular
frame, some of the correlations between the individual fitting parameters are
taken into account.
In the exhaustive search method, one systematically alters the ith fitting
parameter at a series of values and performs a complete nonlinear minimiza-
tion, allowing the remaining n–1 parameters to “adjust,” so as to minimize
chi-square. One then records the series of minimum values possible over a
particular range of the ith fitting parameter. This process is schematically
represented in Figure 5.10 as a series of arrows (i.e., minimizations) originating
at regular intervals along the various parameter axes, which subsequently
relax to the region of lowest possible chi-square given this particular value of
the ith parameter. This method of error analysis takes into account all of the
higher order correlations which may exist between a given set of fitting
parameters. This method also provides the maximum possible variation of a
fitting parameter which is consistent with a particular chi-square range.
A flow chart for the exhaustive search process can be written as follows:
1. Denote the fitting parameter vector at the minimum chi-square value
2. Call an F-statistic-generating routine. (92) This routine, when given a

specific confidence level, degrees of freedom in the analysis, and
will calculate a which represents a value greater than
which one should use to determine when one has searched out “far
enough” along a particular axis.
3. Initialize delta to some small value [e.g., delta = std. errorj (see
above)]:
4. Perform nonlinear analysis of all of the remaining fitting paramters

except with the element fixed at the above value. Set equal
to the minimum value of this particular fit.
5. If (within a specified number of decimal places), then
output the current value of and stop. This value of the jth
fitting parameter will represent the farthest value that this parameter
†
For clarity, only the negative “P” and “Q” exhaustive search calculations are shown in
Figure 5.10.
could take and still be consistent with the data at a particular

confidence level. If is still less than then increase the
delta parameter increment and go to step 3.
The methodology requires a whole series of nonlinear minimizations to be

performed. It is also very easy to implement and is the method currently
utilized by the global analysis program at the LFD. This methodology will
also establish whether a particular model is identifiable or not. For noniden-
tifiable models, the error surface will be completely flat along a particular
set of fitting parameters. Note that the error bars recovered from this type of
error analysis may certainly be nonsymmetric (i.e., not a simple representa-
tion). A comparison of the error bars obtained using the linear approximation
and the exhaustive search approach is shown in Figure 5.11. It is well known
that the uncertainty in a recovered rotational correlation will be at a minimum
when the rotational correlation time has the same value as the total-intensity
decay lifetime. In Figure 5.11, this result is also found numerically. As the
ratio of rotational rate to fluorescence lifetime becomes large the
uncertainty in the recovered should also become very large. With linearly
approximated error, this increased uncertainty occurs very slowly. The more
rigorous error analysis, however, reveals a much more realistic representation
of the actual uncertainties in the recovered rotational correlation time. Note
also that the recovered error bars are very asymmetric, in exactly the manner
which would be predicted: when the positive confidence interval is very
large whereas the negative confidence interval is still bounded; when
the exact opposite occurs. This result reveals that exhaustive search error
analysis can not only assist one in determining a realistic representation
of the uncertainties in the recovered parameters, but can also assist in
experimental design. Although this example is rather obvious (and well
known), there may certainly be cases in which it is not at all obvious which
set of independent variables will be important to alter in order to reduce the
error in a given set of parameters. Examination of the asymmetry in the
recovered fitting parameters can often reveal clues concerning which of these
independent variables are important.
5.8. Conclusions
One of the chief “complaints” concerning the use of global analysis is that
it is model dependent. This is certainly true, and application of a physical
model which is inappropriate for a given data type will certainly yield
questionable results. However, as far as the analysis is concerned, the global
application of a particular model over a multidimensional data surface is as
statistically rigorous a test of a particular model as can be performed. If the

applied model appears to be consistent with a given data surface (all of the
error criteria are satisfied), then there is nothing additional that can be done
to test this particular model. If there are two (or more) physical models which
appear to be appropriate for a given data surface, and both of them have been
successfully applied to the data in a global manner, then one must conclude
that there is not enough information in this particular data surface to dis-
criminate between these two models. To discriminate between these two
models, the global analysis programs (as described in this chapter) allow the
researcher to easily examine, in a quantitative manner, new experimental axes,
which may be capable of discriminating between these alternative models.
These additional axes can be immediately incorporated into the analysis of
the (ever-growing) data set, until (hopefully) only a single model prevails.
How does the global analysis approach relate to other numerical analysis
techniques, such as the application of singular-value decomposition (SVD) to
the analysis of data of the form F(x, y)? A classic application of this type of
analysis is when x = time, and y = excitation or emission wavelength. SVD
acts to separate the spectral- and time-dependent parts of the data matrix.
The spectra are decomposed into the minimum number of independent species
(very useful information). The time-dependent part (a much smaller data set),
now separated from the rest of the data matrix, can be analyzed using
standard nonlinear least-squares techniques. This data reduction technique
has shown itself to be valuable for this type of data. The global analysis
technique, however, can operate in exactly the same manner. With the global
analysis, the time and spectral parts of the problem are not decomposed; they
are simply determined simultaneously in a single analysis. The SVD approach
for this type of data may certainly be less computationally intensive, but the
net result of both types of analysis should (in theory) be identical. The real
difference between the SVD and global analysis approach results when higher
dimensional data surfaces are to be examined.
Data consisting of more than two dimensions cannot be operated on in
a single step using the SVD approach. If the fluorescence data obtained along
the various experimental axes all appear linearly (in the observed data),
various multilinear models can be applied.(93) These types of analyses are in
a very early stage of development (as applied to fluorescence data), yet they
do have the potential of being very valuable methods of analysis. In a global
nonlinear analysis, however, one can analyze as many dimensions as desired
in a single analysis, regardless of whether they relate linearly to the observed
fluorescence or not. Just as importantly, since the analysis is being performed
on the original data surface, there are no problems concerning how to
properly weight the data in the direct global analysis.
Therefore, one should view some of the alternative schemes (e.g., SVD,
factor analysis, multilinear analysis, etc.) as useful analysis tools for particular
data types. By specializing in particular data types, one may obtain both a
reduction in computational time and, in some cases, additional information
which might not be immediately apparent when performing a global analysis.
The global analysis methodology, as described in this chapter, does not have
any qualifiers attached to the types of data which can be combined or the
models which can be applied (other than those specific to all nonlinear least-
squares analyses). As such, it is a general-purpose analysis methodology,
which can be applied to a wide variety of fluorescence data. These features
make global analysis, with the “target” approach, an ideal vehicle for testing
models and recovering fitting parameters. An important advantage of the
numerical procedures discussed here is that the original data are tested
directly against the final model of interest. Multistep analysis is completely
eliminated.
Do the global analysis procedures described here provide a “black box”
that can always be used to obtain system parameters of interest? Certainly
this is not the case. A reasonably good global fit over a large numbr of
different experimental data sets may obscure a poor “local” fit for a particular
experiment(s). The regions where a global fit is not satisfactory can provide as
much useful information as those areas in which the fits are very good. These
regions should not be overlooked. Once trivial instrumental artifacts are
eliminated as a source of the mismatch, the globally applied models should be
reevaluated. The ability to disprove a particular model is often as important
as the determination of the recovered fitting parameters.
The exploration and development of new physical models (and theories)
which will allow additional sets of overdetermination axes to be used in a
global analysis is an important direction for future application. Determining
the “information content” of these high-dimension data surfaces using the
identifiability approach may help in establishing which sets of experimental
axes are worth investigating. The application of the widest possible variety of
physical models to the highest dimensional data set that can be obtained still
provides the experimenter with the best overall chance of understanding the
observed fluorescent system.
Acknowledgments
Much of the inspiration and many of the ideas in this work evolved
through interactions with scientists who have spent time in (or around) the
laboratories of Ludwig Brand and Enrico Gratton. These scientists include
(from the Brand lab) Drs. D. W. Walbridge, R. P. DeToma, G. Ackers,
B. Turner, L. Davenport, R. Dale, M. Barkley, A. Kowalczyk, J. B. A. Ross,
W. W. Laws, M. K. Han, and M. Pritt and (from the Gratton lab) Drs. R.
Alcala, C. Royer, D. Piston, B. Feddersen, W. W. Mantulin, and N. Silva. We

thank Julie Butzow (of the LFD) for expert assistance in figure preparation.
JMB gratefully acknowledges support from the Lucille P. Markey
foundation. JMB is a Lucille P. Markey scholar in biomedical science. LB is
supported by NIH grant GM 11632. Laboratory for Fluorescence Dynamics
(LFD) is supported jointly by the Division of Research Resources of the
National Institutes of Health (RR03155-01) and the University of Illinois
Urbana–Champaign.
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6
Fluorescence Polarization from

Oriented Systems
Thomas P. Burghardt and Katalin Ajtai
6.1. Overview
The polarized emission from dipolar emitters rigidly embedded in the

organized molecular components of an assembled system can indicate some of
the lower angular resolution features of the emitter angular probability density.
This information is useful to indicate changes in the angular probability
density (referred to from now on as N) as the result of perturbations.(1) It is
generally less useful in ascertaining a detailed shape of N due to the limited
amount of angular information provided by dipolar emission, although this
shape determination can be accomplished under some circumstances.(2) We
summarize in this review a general method to quantitate N in terms of an
infinite set of irreducible order parameters. We show that the number and
rank of the order parameters. We show that the number and rank of the order
parameters that are detectable with a particular experimental form of the
polarized emission technique indicate the extent of the angular resolution
theoretically obtainable by the technique.
The experimental techniques discussed in this chapter include time-
resolved fluorescence depolarization from mobile components of a biological
assembly and steady-state fluorescence polarization from immobilized or
slowly moving components of a biological assembly. The time-resolved
technique detects higher resolution features of N. A closely related technique,
electron spin resonance (ESR) of spin labels in ordered assemblies, detects the
spin angular probability density with a resolution that exceeds that obtainable
from fluorescence techniques. The mathematical relationship between ESR-
and fluorescence polarization-detected angular densities is discussed, and an
Thomas P. Burghardt and Katalin Ajtai • Department of Biochemistry and Molecular
Biology, Mayo Foundation, Rochester, Minnesota 55905. K. Ajtai was on leave from the
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary. (Manuscript
submitted January 1, 1988.)
307
308 Thomas P. Burghardt and Katalin Ajtai
example using ESR data to supplement information from fluorescence

polarization is described.
The extraction of useful information from an emitted signal depends
ultimately on the use of an appropriate probe molecule. The biochemical
methods for placing fluorescent probes specifically and rigidly on molecular
components of biological assemblies are well developed. Equally important,
biochemical methods for verifying the position and specificity of the probes
are also elaborate. We summarize the principles of these methods to give
some idea of the biochemical difficulties associated with useful probe studies.
In each of the subsections of the main body of this chapter (Section 6.2),
we have included examples, usually describing the experimental realization of
the ideas presented in the subsections. In this way we wish to convey the
message that our general approach to the investigation of angular order in
biological assemblies is practical. Although all of the examples are taken from
probe studies of muscle fibers, reference is made to other systems of interest
such as lipid membranes or supported lipid monolaye-rs.
The investigation of angular order in muscle fibers is the focus of
the applications discussed in this chapter (several good reviews of muscle
structure exist (3–5) ). The muscle fiber consists of two interdigitated protein
filaments that interact to produce fiber shortening during muscle contraction.
The thick filament (named for its appearance under the electron microscope)
is composed of spatially assembled dimers of myosin proteins, and the thin
filament is two strands of polymerized actin proteins wound in an -helix. The
myosin molecules contain a globular enzymatic (ATPase) head region that
interacts cyclically with an opposing thin filament while hydrolyzing ATP
during fiber shortening. Since a model for this interaction of the myosin head
with the actin was first proposed to involve a rotation of the head moiety
while attached to actin, (6,7) intensive study of the angular disposition of the
head has occurred. The careful determination of the angular disposition of the
head is clearly an important experimental objective, and the reliable data now
published are ambiguous enough to have caused the production of a confusion
of data interpretations. This ambiguity is likely due in part to the complexity
of the system and more importantly to the resolution limitations imposed by
the experimental techniques employed. This circumstance in muscle research
caused us to undertake the systematic investigation of the angular resolution
in the fluorescence techniques and to develop both methods to reduce data
ambiguities as well as a unified approach to the description of N that would
allow relevant data from different techniques to be compared.
6.2. Theory and Application

The theoretical description of angular order of components of a biological
assembly is centered on a model-independent approach in which we expand
Fluorescence Polarization from Oriented Systems 309
the angular probability density in terms of an appropriate set of complete

angular functions with unknown expansion coefficients. In Section 6.2.1.
we discuss this idea in detail. In Sections 6.2.2 and 6.2.3 we discuss some
experimental fluorescence techniques for measuring the angular probability
density, N. In Section 6.2.4 we compare the fluorescence techniques with ESR
techniques for measuring N and show an example of the use of ESR data in
combination with fluorescence data to form a more accurate description of N.
In Section 6.2.5 we briefly discuss the biochemical techniques for attaching
covalent probes to the components of the biological assembly.
6.2.1. The Angular Probability Density N
The probability density function N describes the angular distribution of

a particular set of molecule-fixed coordinate frames in a biological assembly
of molecular components, relative to a laboratory-fixed coordinate frame. Any
molecular frame is related to the laboratory frame by a rotation. We choose
to describe this relationship using the Euler angles and which are
denoted collectively by It is convenient to expand in terms of Wigner
functions that form a complete, orthogonal basis set of functions on
the domain and denoted by such that
The parameter is called an order parameter of rank j, and it represents

the contribution to N of the (j, m, n)th orthogonal component of the set of
basis functions. The individual are complex and can be negative. N is
real and is greater than or equal to 0 as required for it to have meaning as
a probability density.
The ’s are orthogonal on such that
where * indicates the complex conjugate, is the Kronecker delta, and

is the integration element The orthogonality property of
Eq. (6.2) shows the normalization of N, such that implies
The order parameters are unknowns that we measure experimen-

tally. The rank of is given by j. The angular resolution of an experimental
method is theoretically limited by the rank and total number of the order
parameters detected. Consider, as an example, a system in which all of
310 ThomasP. Burghardt and Katalin Ajtai
the molecular frames have the unique polar angle and are randomly
distributed in the other Euler angles and The normalized angular
probability density for such a system is
where is a Dirac delta function. Equation (6.3) is inverted using

Eq. (6.2), and the order parameters of this probability density are easily
shown to be
where is a Legendre polynomial. Shown in Figure 6.1 is the probability

density computed using Eqs. (6.1) and (6.4) for and including in
the summation of Eq. (6.1) the order parameters and (the limit
of resolution typical for fluorescence polarization; see Section 6.2.2). For
comparison, we also show a probability density computed by including the
order parameters and (resolution more typical of
electron spin resonance) in the summation of Eq. (6.1). The plots demonstrate
both the best estimate obtainable by fluorescence polarization when describing
the probability density of Eq. (6.3) and the relationship between angular
resolution and order parameter rank.
In the next section we derive the relationship between the order
parameters and the measurable fluorescence polarization signal.
6.2.2. Fluorescence Polarization in Homogeneous Space
In fluorescence polarization experiments a fluorescent probe is specifically

and rigidly fixed in the molecular frame of a component of the biological
assembly. A propagating, linearly polarized, electromagnetic field excites the
probe. The fluorescent emission efficiency is detected as a function of one or
more parameters such as the polarization of the excitation or emission fields.
The probability of a single probe absorbing light and subsequently emitting
the energy as fluorescence is denoted by K. K depends on the molecular frame
Euler angles and can be expanded in terms of the Wigner functions just as
we did for N in the last section. We find
where the coefficients are calculated from the well-known expression

describing the interaction of a fluorophore with an electromagnetic field (see
below). The fluorescence signal, F, for an ensemble of probes is given by
Substituting Eqs. (6.1) and (6.5) into Eq. (6.6) and using Eq. (6.2), we find
With known, a linear combination of the is determined by observing F.

The expression for K appropriate for a probe in homogeneous space is
explicitly
where c is a constant, and are the unit electric dipole moments of the
fluorophore for absorption and emission, respectively, E is the electric field
vector of the excitation light, and v is the unit vector of polarization of the
emitted light that is observed (using a polarizer).
The are calculated using Eqs. (6.2), (6.5), and (6.8) and then
substituted into Eq. (6.7), and the summation is carried out. This calculation
of F, using Eq. (6.7), is straightforward but tedious and has been done for the
general case, including corrections for high-aperture optics.(8) The reader
should consult Ref. 8 for a slightly more detailed description of the calcula-
tion. An important and general result for fluorescence polarization, however,
is derived in this calculation where it is shown that the summation on j
for is limited to This result holds generally for fluorescence
polarization in homogeneous space and, as discussed in Section 6.2.1, sets
a theoretical upper limit on the angular resolution obtainable from the
technique.
We performed the summation of Eq. (6.7) for specific applications using
a symbolic manipulation computer program (SMP, Inference Corp.,
Pasadena, California). This latter procedure is more convenient for all but the
most simplified applications for the analysis of fluorecence polarization data.
Below we apply this formalism to the study of
ethylenediamine (l,5-IAEDANS)-labeled myosin cross-bridges in
muscle fibers.
6.2.2.1. Example 1: Fluorescence Polarization from 1,5-IAEDANS-Labeled

Muscle Fibers
Muscle fibers are made up of actin and myosin filaments that slide in
opposite directions during muscle shortening. The myosin filament is
composed of myosin molecules that have a globular head region that can be
proteolytically cut from the rest of the molecule (subfragment-1 or S-l). The
S-l moiety spans the distance from the myosin to actin filaments and during
muscle shortening is the site of the ATPase activity. A proposed model of
muscle contraction holds that the S-l rotates while bound to the actin to
produce the filament sliding. Because of this model it is of interest to muscle
researchers to determine the angular probability density of S-l in the fiber
when the fiber is subjected to various chemical and mechanical perturbations.
In this example single muscle fibers were washed in a buffer solution
and transferred to a quartz slide on the microscope stage. Extrinsic S-l,
which binds rigidly to the actin filaments in the fiber at bare zones where the
intrinsic myosin is not present, was specifically labeled with the fluorescent
probe 1,5-IAEDANS and allowed to diffuse into the single muscle fibers.
The experimental apparatus shown in Figure 6.2 consists of a lens that
focuses an argon ion laser beam on the back image plane of the
epi-illumination microscope holding the muscle fiber. (2) All of the optics in
the excitation light path were quartz to minimize background fluorescence.
A Pockels cell rotates the laser polarization through 90° every 5 ms such that
the polarization rotates from parallel to perpendicular with respect to the fiber
axis. The polarized fluorescence emission is collected in intervals synchronized

with the laser polarization and quantitated by photon-counting electronics.
The filter block (see Figure 6.2) contains a dichroic mirror and a barrier
filter. The dichroic mirror reflects the excitation light into the objective
and transmits the longer wavelength emission collected by the objective.
A rotatable film polarizer analyzes the polarized emission.
Known properties of the muscle fiber and a particular choice of the
molecular frame greatly reduce the number of order parameters on which an
appropriate angular probability density can depend. There is experimental
evidence indicating that a muscle fiber has azimuthal symmetry such that it
is unchanged by an arbitrary rotation about the fiber axis.(1) If the fiber is
placed lengthwise along the lab frame z axis, then is the independent
variable for the probe angular probability density about the fiber axis, and
azimuthal symmetry in the fiber requires N to be independent of This fact
is expressed implicitly in N by requiring in Eq. (6.1). It is well

known that a 180° rotation relates opposite half-sarcomeres in the muscle
fibers. This symmetry property requires unless j–n = 0, 2, 4, .... Further-
more, we choose to express the probe angular distribution relative to a
molecular coordinate frame wherein the absorption dipole, , points along
the z axis, and the projection in the x–y plane of the emission dipole,
points along the x axis. This choice of molecular coordinate frames makes
the polar angle and the torsional degree of freedom of the molecular frame
and renders the fluorescence polarization signal independent of order
parameters except when n = 0, ±2 (inspection of Eq. 17 in Ref. 8 verifies
this result). This last restriction on n is not a result of fiber symmetry but of
the properties of dipolar emission.
The theoretical fluorescence intensity, F, computed from Eq, (6.7) for the
microscopic application was shown to be(2)
where is the laser polarization and the emission polarization as shown in

Figure 6.3, is a constant, and are the correction factors for
high-aperture collection of polarized light, (9) and
The quantities appearing in Eqs. (6.12)–(6.14) are

Clebsch–Gordon coefficients,(10) and the parameter in Eqs. (6.13)–(6.17)

is the polar angle of the emission dipole in the molecular frame. All other
quantities in the sum of Eq. (6.9) that are not explicitly mentioned in
Eqs. (6.10)–(6.17) are zero.
A fluorescence polarization experiment consists of the measurement of
four intensities denoted by The symbols and
mean parallel and perpendicular to the fiber axis while the first index on F
refers to excitation polarization and the second to emission polarization.
Three ratios, defined below by Eqs. (6.18)–(6.20), combine the four intensities
into the quantities , which are independent of the absolute
intensity calibration of the optical instrument. The normalized ratios are
defined by
To solve Eqs. (6.18)–(6.20) for the unknown order parameters, we must also
know the optical correction factors and and the emission dipole
polar angle, The correction factors are computed from formulas derived
previously (see Eq. 20 in Ref. 9) and are and
(for glycerol immersion quartz objective with a numerical aperture
of 1.25 and quartz interfaces). The angle between the absorption and emission
dipoles was estimated by measuring from 1,5-IAEDANS-labeled S-l in
buffer + 50% glycerol (1:1 buffer to glycerol by volume, pH 7.0) solution
cooled to –20°C and solving Eq. (6.9) with We
found for the excitation wavelength of 364 nm and filter block cutoff
wavelength at 420 nm that
The restrictions on the order parameters for muscle fibers, shown pre-
viously to require with j = 0, 2, and 4 and n = 0, cause
Eqs. (6.18)–(6.20) to depend only on the four parameters
and Because there are four unknowns with three
equations to constrain them, we must supply one new constraint. We show in
Section 6.2.4 that we can obtain the additional constraint from the model-
independent analysis of ESR spectra from muscle fibers.(2)
Fluorescence polarization experiments on 1,5-IAEDANS-labeled S-l
bound to muscle fibers were performed on two states of the muscle fiber. One
state, called MgADP, occurs when the fiber is bathed in buffer containing
MgADP. In this state both the nucleotide and actin are bound to S-l. The
second state, rigor, occurs when the nucleotide is removed. With the help of
the ESR data, as described in Section 6.2.4, we find for rigor fibers:
We summarize the results with a plot of the polar angular probability

density found from by averaging over the Euler angles That is,
Plots of for fibers in rigor and in the presence of MgADP are shown in
Figure 6.4. These plots of the probe orientation probability density indicate
that the probe rotates upon the binding of MgADP. To ascertain if this result
also indicates that S-l rotates upon MgADP binding, we must undertake
control experiments to determine whether or not the probe remains rigidly
fixed in the S-l under these conditions. Generally, two time-resolved fluores-
cence experiments are helpful as controls. First, the probe lifetime is measured
under appropriate conditions since the lifetime is often environment sensitive
and may change if the probe rotates locally.(11, 12) Second, if possible, the time-
resolved fluorescence depolarization from the labeled, isolated protein element
from the ordered ensemble (S-l in this case) tumbling by Brownian motion
in solution is measured. This measurement senses motion of the probe or
a domain of the protein containing the probe, if the motion is a rotation
that changes the probe orientation relative to the hydrodynamic frame of
the protein element.(13, 14) In this experiment (in agreement with previous
studies (15, 16 ) ) both controls indicate that the 1,5-IAEDANS remained fixed in
the hydrodynamic frame of S-l in the presence and absence of MgADP,
suggesting that the probe rotation observed with fluorescence polarization
was due to S-1 rotation.
6.2.2.2. Example 2: Wavelength-Dependent Fluorescence Polarization

Fluorescence polarization studies (as in Section 6.2.2.1) established the
ability of the myosin cross-bridge (the S-l moiety of myosin in a muscle fiber)
to bind to the actin filament at more than a single orientation.(1, 17, 18) This
observation is based on experiments using a single probe, iodoacetamido-
tetramethylrhodamine (IATR), covalently linked to cysteine 707 (also called
sulfhydryl 1 or SH 1 ) or myosin. The IATR probe showed that the cross-
bridge maintains one orientation in rigor and a second orientation when
the cross-bridge binds MgADP. Spin probes (19, 20) and 1,5-IAEDANS (17)
also linked to SH 1 , showed no angular reorientation upon the binding of
MgADP. The studies of Borejdo et al.(17) on the 1,5-IAEDANS probe were
performed before the studies described in Section 6.2.2.1 and with the lower
resolution technique linear dichroism of fluorescence.
These conflicting findings obtained with IATR on the one hand and with
1,5-IAEDANS and spin probes on the other suggested that the probes have
differing orientations in the S-1 molecular frame such that rhodamine is poised
on the cross-bridge at an angle favorable for the detection of cross-bridge
angular displacement while 1,5-IAEDANS and the spin probes maintain
an unfavorable orientation.(21) With wavelength-dependent fluorescence
polarization, we use the ability to change the direction of the 1,5-IAEDANS
absorption dipole by changing the excitation wavelength (22) to directly
ascertain if differing dipole orientations could account for this variability of
results.
The wavelength-dependent experiments were performed on a commercial
fluorescence spectrometer with Glan–Thompson polarizers in the excitation
and emission beam paths. A rectangular stainless steel support, made to fit in
a standard fluorescence cuvette, held mounted fibers in the optical paths (see
Figure 6.5). The muscle fibers were mounted in the vertical configuration such
that the fiber axis was perpendicular to the plane defined by the path of the
excitation and collected emission beams as in Figure 6.5. As in the example
described in Section 6.2.2.1 S-l was specifically labeled with 1,5-IAEDANS
and allowed to diffuse into the muscle fiber under rigor conditions. After ~ 30
min the free labeled S-l was washed out with rigor buffer, and the experiment
was performed on the fiber decorated by S-1.
In this application we measured [see Eq. (6.19)] as a function of
excitation wavelength, The results, shown in Figure 6.6, indicate a region
in the spectrum where for fibers in the presence of
MgADP and for fibers in rigor. (2) For a random distribution of probes
at any These data indicate that the probe angular probability
densities for these two states are unmistakably different since the addition of
a random probability density to either probe density will not transform one
into the other. As described in Section 6.2.2.1, the controls indicate that the
1,5-IAEDANS remained fixed in the hydrodynamic frame of S-l in the
presence and absence of MgADP, suggesting that the probe rotation observed
was due to S-1 rotation.
6.2.3. Time-Resolved Fluorescence Depolarization Determination of

the High-Resolution Angular Probability Density
Time-resolved fluorescence depolarization following polarized excitation

is generally used to study the nanosecond rotational motion of molecules in
solution. Several investigators also employed the technique to look at
nanosecond motion of elementary subunits of biological assemblies. The
application of this method to biological assemblies yields information related
to the time-averaged angular probability density, N, that augments the
information obtained from the time-independent fluorescence polarization
methods discussed in the last section.
The calculation of the time course of the emission decay from rotational
motion of a fluorescent (labeled) protein element of a biological assembly can
be done using a suitable model. Kinosita et al.(23) developed a model theory
for fluorescence polarization decay that was particularly suitable for lipid
membrane applications. The model theory, generally characterized as
rotational diffusion in an angular potential, was subsequently generalized
to include more elaborate angular potential functions.(11, 24–27) Prior to this
fluorescence work, closely related calculations were performed to determine
the effect of probe rotational diffusion in a potential on the shape of ESR
spectra.(28–30) We have subsequently introduced a model-independent approach
to this problem that, among other attributes, demonstrates the contribution
of the time-dependent signal to the determination of the time-averaged
angular probability density, N.(31) We summarize the model-independent
approach and describe a biological application below.
The equation describing molecular rotational motion is assumed to be
where t is time, H is an operator, and is the time-dependent prob-

ability density for molecular orientations. If H is not explicitly dependent on
time, Eq. (6.30) is separable in time and space coordinates so that
is the solution to Eq. (6.30) when The initial condition, determined by

the experimental application, is expressed in the expanded form
where is a constant coefficient. Equation (6.32) can be inverted using

Eq. (6.2) to give
In a time-resolved fluorescence depolarization experiment, the intensity

emitted by fluorophores specifically attached to elements of the biological
assembly is observed. The fluorescence intensity that a fluorophore with emis-
sion dipole emits into a polarizer oriented with its polarizing axis along v
is . The fluorescence intensity from an ensemble of such fluorophores,
F(t), is given by
where G(t) contains the time dependence of the signal due to the fluorescence
lifetime of the probe. The explicit computation of F(t) using Eq. (6.34) was
described in detail previously.(31) This calculation is performed using
Eqs. (6.2) and (6.32) and the expression for in terms of Wigner
functions that has been derived in several papers (e.g., Ref. 14). We find
and
where and are spherical unit vectors along the x, y, and z axes in
the laboratory frame. T is the time development operator relating and
through the linear operation such that
with matrix elements given by
In further discussions we assume that the fluorescence decay function G ( t ) is

proportional to a single exponential with half-time When
where is the maximum rotational relaxation rate of the ordered
system, it is appropriate to expand in Laguerre polynomials,
such that (32)
The functions defined by
so that we can express the fluorescence signal following polarized excitation

by a pulse of infinitesimal duration, F(t), as a sum of orthonormal functions
of time given by
where
and
The coefficients in Eq. (6.44) are determined by the initial condition

as given by Eq. (6.33). For the usual case of initial excitation by a
short pulse of polarized light, discussed in the following example, we have
where N is the steady-state angular probability density and satisfies the

steady-state condition From the general expression for N in
and
Eq. (6.1),
Eq. (6.33),
the expansion
we solve for
of in Wigner functions shown elsewhere,(31)
We notice from Eq. (6.47) that the parameters depend on (order

parameters) of all ranks. Some of the order parameters can be determined by
time-independent methods as described in Section 6.2.2. Our purpose in
describing this method here is to show how it can approximate order
parameters of ranks not detected by the (standard) time-independent techni-
ques (as in Section 6.2.2). This is done by solving for the coefficients in
Eq. (6.47) from the time-resolved signal that is related to the via
Eq. (6.44). The are related to the order parameters by Eq. (6.47).
We discuss an application of the time-resolved method that enabled the
calculation of rank 6 order parameters in Section 6.2.3.2 below.
With Eqs. (6.43)–(6.47) the time-resolved fluorescence depolarization
signal is constructed in a model-independent manner. The unknown
parameters are generally the matrix elements of the operator The linear
combinations of these matrix elements that are the coefficients in Eq. (6.44)
are determined experimentally from the time-resolved curves.
6.2.3.1. Considerations for Real Instruments and Related Numerical Methods

On a real instrument the excitation lamp pulse has a finite width that is
convoluted with the fluorescence emission signal F(t). It could be essential to
account for the finite width of the lamp pulse when the fluorescence lifetime
of the probe is much shorter than any of the motional relaxation times of
the ordered systems Under these circumstances the
observed signal, F(t), is ( 3 l )
where is the shape of the lamp pulse. Using Eq. (6.43), we find
or
where
is the new function of time in which the observed fluorescent signal is

expanded. Unlike the of Eq. (6.41), the are not orthogonal. The
departure of the from orthogonality depends on the shape of the lamp pulse
such that when the pulse is infinitesimal in duration, that is, it is a function
in time, Using a general numerical method described for an ESR
application, we calculate the coefficients from F(t). The procedure is
described briefly below.
We define a projection operator by the equation
where is a constant independent of time. We require that have the

property
Then,
We construct the operator using the constants which are found by

substituting Eq. (6.52) into Eq. (6.53) and solving for We find
where is the (i, j)th element of the inverse of the matrix

with elements This method was used in both the ESR and
time-resolved fluorescence applications.
6.2.3.2. Example 3: Application to Fluorescent-Labeled Muscle Fibers

We measured the time-resolved fluorescence depolarization from
1,5-IAEDANS-labeled cross-bridges in muscle fibers in the presence of ATP
without In this state, called relaxed, the myosin cross-bridges are
known to be in a rapid equilibrium between actin-attached and detached
states(34, 35) so that the fiber can be slowly stretched with the minimum of
resistance. Borejdo and Putnam (36) showed that the myosin cross-bridge was
selectively labeled with 1,5-IAEDANS, using a method developed by Duke
et al.(37), such that of the label was covalently linked to Nihei
et al.(38) showed that modification of with 1,5-IAEDANS does not impair
fiber contractility when the degree of labeling is as high as 0.8 mole of
fluorophore per mole of myosin, implying that the modified cross-bridge is

able to produce force in the same manner as the native cross-bridge.
In this example we employed low-aperture excitation and collection
optics so that the excitation electric field, E, is linearly polarized and
We choose E to be polarized along the laboratory frame z axis
so that E = (0, 0, 1). We collect the emission polarized along the laboratory
frame z and x axes [corresponding to and in Eq. (6.37)]. The
observed signals are then
When This approximation is appropriate for

the muscle fiber application.
The parameters in Eqs. (6.56) and (6.57) are the following: the coeffi-
cients which depend on the order parameters and the absorption
dipole moment , the emission dipole moment and the matrix
elements of The values of and some of the can be determined
from independent observations. Mendelson et al.(39) estimated the directions
of and for myosin cross-bridges modified at by 1,5-IAEDANS. The
order parameters are dependent on the physiological state of the muscle, and
some of them are known for relaxed cross-bridges.(27) Usually only the matrix
elements of in Eqs. (6.56) and (6.57) are unknowns to be determined in the
time-resolved experiment.
The time-resolved data give supplemental information related to the
steady-state angular probability density, N, when we make use of a particular
model for cross-bridge motion. When the operator H describes a probe
moving with rotational Brownian motion in a potential, then
where is the rotational diffusion constant, is a differential operator

identical to the quantum-mechanical orbital angular momentum operator,

and f is related to N by
with related to the angular potential V by
where k is Boltzmann’s constant, and T is temperature.

The matrix elements of H are calculated using Eq. (6.2) and the relations
from which we find

where
Higher order matrix elements are calculated from Eq. (6.65) using the com-
pleteness of the Wigner functions on the interval . Completeness requires
where is a Dirac function. With Eq. (6.67) we can show that the
second-order matrix element, is
Higher order matrix elements can be similarly constructed.

Time-resolved measurements were made on muscle fibers in two
configurations. In the vertical configuration the fiber axis (the z axis in the
laboratory frame) is parallel to the excitation light polarization so that the
depolarization curves are sensitive to rotational motions of the probes about
axes other than the fiber axis. In the horizontal configuration the fiber is
rotated by 90° so that the excitation light polarization is perpendicular to the
fiber axis, and its light propagation vector makes an angle of 45° with the
fiber axis (see Figure 6.5). Depolarization curves from the horizontal fibers are
sensitive to probe motion about axes parallel to the fiber axis. By requiring
our model of Brownian motion in an angular potential to account for the
relaxation rates of the cross-bridges in both fiber configurations, we acquire
sufficient constraints on the free parameters to estimate the rank 6 order
parameters of N.
With the available steady-state fluorescence polarization data,(40) we
showed that in the vertical fiber configuration, N is described by the order
parameters
For the horizontal fiber configuration we can readily compute the new
horizontal order parameters, denoted by from the vertical order
parameters, using the relation
A similar relation holds for in Eq. (6.66). With the rank order
parameters given as known quantities, we estimate the rank 6 order
parameters using the model.
An interesting new feature of the proposed polar steady-state angular
probability density computed using Eq. (6.29) is introduced by the addition
of the rank 6 order parameters. Shown in Figure 6.7 is a comparison of proba-
bility densities with and without the inclusion of the rank 6 parameters. The
higher resolution plot has a bimodal feature not observed at lower resolution.
6.2.3.3. Generalized Method for Evaluating Matrix Elements of T

The expansion of in terms of Laguerre polynomials, done in
Eq. (6.40), is not easily extended to the case in which the rotational relaxation
half-times of the system are similar to or smaller than the probe lifetime. We
propose here an untried method that makes use of Laplace transforms to
isolate the lowest order relaxation rates from the time-resolved fluorescence
data. We also make use of some new notation to make the derivation simpler.
Let the ordered triplet of indices (j, m, n) from be summarized by
a single index i such that is equivalent to Then we can write
Eq. (6.35) in the form
where summarizes the time-independent constants. Again assuming G(t) to

be a single exponential,
If we can neglect the width of the lamp pulse, then the observed signal is given
by Eq. (6.71). F(t) is Laplace transformed to give the quantity such that
so that
Equation (6.73) can be expanded in powers of to give

to lowest order in H,
On a plot of as a function of is the y-intercept, and the slope

is a function of the first-order matrix elements of H.
6.2.4. Relation of Electron Spin Resonance Spectra to Fluorescence Polarization
6.2.4.1. Rotation Connecting Spin and Fluorescent Probe Order Parameters

There are many well-developed methods available that use ESR spectra
to determine spin probe angular probability densities.(42,43) As mentioned
in Section 6.2.2, we made use of ESR data in the determination of order
parameters of the angular distribution of 1,5-IAEDANS-labeled cross-bridges
in muscle fibers in example 1. In example 1 we described the determination of
the four unknown order parameters,
from the three quantities measured by fluorescence polar-
ization [see Eqs. (6.18)–(6.20)] with additional information from the ESR
spectrum. In this section we wish to describe in more detail the connection of
the ESR and fluorescence polarization techniques for determining order.
The fluorescence and ESR data are quantitated in terms of order
parameters, but the values of the order parameters are not interchangeable
because in these applications both sets of order parameters describe the probe
angular probability density. The probe angular probability density derived
from ESR data describes how the spin probe is oriented. Likewise, the probe
probability density derived from fluorescence data describes how the fluores-
cent probe is oriented. A rotation can always be found to relate the two sets
of order parameters such that (2,8)
where represents the Euler angles describing the rotation

from the spin probe fixed frame to the fluorophore fixed frame. Equation
(6.75) transforms the order parameters calculated in the principal magnetic
frame of the spin probe to the reference frame of the fluorescent probe (for
example, our choice with the z axis along the absorption dipole and the x axis
along the projection of the emission dipole in the x–y plane). Known sym-
metry properties of the muscle fiber simplify Eq. (6.75), reducing it to the
expression
We estimate by initially guessing its value and then calculating

and (fluorescence) using Eq. (6.76) and known values for
(see, for example, Ref. 33). We then solve Eqs. (6.18)–(6.20) for
and (fluorescence) and compare these values with the values of
and (fluorescence) computed directly from Eq. (6.76) and (ESR).
On the basis of this comparison, an improved estimate of is made, and
we repeat the above steps until the difference between the fluorescence order
parameters computed by the two methods is minimized. By this method, infor-
mation from ESR can augment fluorescence data to provide a more complete
description of the fluorescent probe angular probability density.
6.2.4.2. Example 4: Fluorescence Polarization from 1,5-IAEDANS-Labeled

Muscle Fibers (Example 1, Continued)
ESR spectra measured from fibers in rigor and in the presence of
MgADP were analyzed by a model-independent method to yield the order
parameters of the spin probe angular probability density.(41) We found
in this investigation that
and for fibers in rigor, and
and
for fibers in the presence of MgADP. By the
method outlined above (Section 6.2.4.1), we estimate and
These values for and (ESR) were used in Eq. (6.76) to
compute the order parameters (fluorescence) listed in Eqs. (6.21)–(6.28).
6.2.5. Biochemical Techniques of Specific Labeling
An important task in the study of oriented systems of biomolecules is to

find a reporter group that fits specifically at a well-defined point on the
biomolecule without altering its biological activity. One can introduce the
fluorescent derivatives specifically using covalent or noncovalent interactions
between the probe and the biomolecule. After the modification, methods for
verifying the location of the probe and for assaying the biological activity of
the modified system must be developed.
6.2.5.1. Covalent Fluorescent Labels

A well-chosen covalent probe reacts specifically with the region of interest
on the target biomolecule.(44) The method of making a specific fluorescent
probe is to synthesize a derivative from the dye by attaching a spacer with a
free reactive group at one end that selectively modifies particular side chains
on the target molecule. For proteins, the most often used probes are the
thiol-selective (iodoacetamido, iodoaceto, maleimido, and bromoacetamido)
or amine-selective (isothiocyanato, carboxyl, sulfonyl acid, and succinimidyl
ester) derivatives. Examples of these probes are drawn in Figure 6.8, where it
is shown that tetramethylrhodamine can be used as both a sulfhydryl- and an
amino-selective probe.
In most cases, the amino acid side chains of the protein that are involved
in the molecular aspects of biological activity have altered reactivities due to
their irregular values. This gives one the possibility to introduce labels
exclusively at these points. By lowering the pH of the reaction medium (to
within pH 6–7) far below the of the majority of the lysine groups
the ionized form of these groups becomes unfavorable for
covalent reactions while cysteinyls, especially those having altered values,
can still react. The sulfhydryl (Cys-707) of myosin is an example of this
phenomenon. reacts specifically with iodoacetamide derivatives of dif-
ferent fluorophores near pH 7 under mild conditions. (45,46) The remaining SH
groups available to the dye react slowly. The reactivity of is so enhanced
that it can serve as a single labeling point in a whole muscle fiber system. (47,48)
Using sulfhydryl-specific labels, we can also change the possible target side
chain by prereacting the system with certain reversible SH-blocking com-
pounds. The reactive thiols are reversibly blocked, and the chemically less
reactive groups are exposed to the covalent by binding probe. After the
covalent binding procedure is completed, the blocking is reversed, restoring
reactive thiols to their native state.(46,49)
The chemical characteristics of the probes themselves also affect the
binding of the label to the target molecule. Rings with apolar character react
preferentially with lipids, membranes, or protein side chains in hydrophobic
crevices. Polar probes label groups on the more hydrophilic surfaces of the
protein.
We see that the reaction of a specific fluorescent probe with a protein
depends on the nature of the reactive group attached to the chromophore
and on the interaction between the dye and the environment of the side
chain to be labeled. The three-dimensional fit of an optical probe also could
be influenced by other factors. The best fit of the chromophore ring on the
protein surface could vary for different stereoisomers of the label, leading to
nonhomogeneous labeling. This would be detected by nonhomogeneity in the
fluorescence lifetime and would cause difficulties in the interpretation of
the polarized fluorescence. The length of the spacer group from the dye to the
reactive group also is an important factor with regard to the specificity and
rigidity of the probe. With short spacers, dyes are introduced at the protein
surface while longer side chains allow the probes to penetrate into proteins
and membranes. This latter property is facilitated by an apolar spacer group.
An example of this is provided by the derivatives of pyrene shown in
Figure 6.9. An increasing number of apolar groups allows the label to
modify points deep inside a folded macromolecule.
6.2.5.2. Noncovalent Labeling Techniques

Proteins and lipids can also be labeled using specific noncovalent associa-
tions. This technique, called affinity labeling, uses ionic, hydrophobic, or
hydrophilic interactions to complex the probe with the biomolecule. The
probe–biomolecule interaction can subsequently be made more permanent
using photoaffinity labeling methods (50) or by probe trapping using cross-
linkers.(51)
The affinity probes are a rather heterogeneous group varying from the
fluorescent lanthanides (which substitute for calcium in calcium-binding
proteins such as actin (52) and calmodulin) to fluorescent derivatives of
nucleotides, coenzymes, enzyme substrate inhibitors, and other proteins.
Fluorescent derivatives of adenine nucleotides were used for orientation
studies in muscle fibers,(53) as was rhodamine-labeled phalloidin, a molecule
that binds very tightly to actin.(54,55) Fluorescent-labeled antibodies provide
a whole class of probes that can be targeted to a particular sequence of
polypeptides with three-dimensional structure.(56)
6.2.5.3. Labeling Methods

The careful fluorescent probe study of oriented biological systems requires
that we introduce the probe while maintaining the native biochemical character
of the system. Usually, fluorescent dyes are introduced in the biological
system at neutral pH, in mild salt buffers. We must be sure that the probe is
solubilized in the labeling buffer and that it can penetrate the membrane
covering the cells or tissues. The large aromatic rings of tetramethylrhodamine
labeling muscle fibers, for example, require that we first dissolve the probe at
high concentration in an apolar organic solvent (dimethylformamide) before
diluting it ~100-fold in the neutral buffer labeling solution. The muscle fiber
is demembranated chemically by bathing it in a mild neutral detergent (Triton
X-100, 0.1–0.2%) before the application of the labeling solution. After the
labeling is completed, care must be taken to remove the unreacted dye to
prevent further (nonspecific) labeling and to ensure that the fluorescence
signal originates from the covalently attached probe. This can be done by
extensive washing in probe-free buffer.
6.2.5.4. Probe Specificity

Once a protocol is worked out that places a sufficient amount of probe
on the biological assembly such that a fluorescent signal with a high signal-to-
noise ratio is detected, the next step is to localize the probe in the system. This
is obviously important when a particular constituent of the system is of
experimental interest and is essential for unambiguous interpretation of the
polarized fluorescence signal. An example of this process is described below.
The skeletal muscle fiber is an ordered molecular assembly of two major
proteins and eight minor proteins (see Figure 6.10). To study the orientation
of the myosin head during changes in the physiological states of the fiber, we
have covalently labeled the thiols on S-l with 4-[N-(iodoacetoxy)ethy1-N-
methyl]amino-7-nitrobenz-2-oxa-l,3-diazole (IANBD). The labeling proce-
dure follows the general procedure of fiber labeling introduced by Borejdo
and Putnam. (36) The labeling is carried out on fibers that have been demem-
branated by chemical skinning with Triton X-100, at 0°C in 100 dye

concentration for 15 min at neutral pH in phosphate buffer. The reaction is
stopped by exhaustive washing of the fiber in probe-free buffer.
The whole protein assembly in the fiber is dissolved in sodium dodecyl
sulfate (SDS), and SDS–polyacrylamide gel electrophoresis (SDS-PAGE)
analysis is performed. The result of this analysis is shown in Figure 6.11. Of
the ten different protein components, only two are fluorescently labeled. As
judged from the fluorescent intensity measured from a scan of the gel, ~82%
of the dye was incorporated into the heavy polypeptide chain of myosin while
18 % of the total fluorescence is associated with the troponin I protein.
The localization of the label in the primary sequence of the myosin is
carried out on myosin purified from labeled muscle fibers. The labeled protein
is proteolytically cut to yield S-l, and further tryptic digestion of S-l yields
three peptides of 20, 50, and 27 kilodaltons (kDa). Following the fluorescence
on the gel as shown in Figure 6.12 allows us to identify the labeled peptide as
the 20-kDa fragment, which contains the two fast-reacting sulfhydryls
(57)
and More specialized methods are required to biochemically separate
and These methods are described elsewhere.(58)
6.2.5.5. Biological Activity

Of equal importance to the localization of the probe is the assessment of
the damage caused by the incorporation of the probe into the biological
system. The native biochemical activities of the system must be essentially
preserved in the probe-modified system for data interpretation to be meaning-
ful. This question can be complicated since some biochemical alteration by the
probes is inevitable, and one must be selective in deciding what deviation
from the native biochemical activities can be tolerated. Again an example

from muscle illustrates this point and is described below.
The enzymatic activities of the myosin cross-bridge are significantly
altered by the probe modification of myosin at It is well known that
upon modification of the ATPase of myosin in the presence of is
activated 2–10-fold while the ATPase in the absence of divalent cations, the
so-called ATPase, is inhibited linearly with the fraction of
modified.(45,46) Surprisingly, the active isometric tension of muscle fibers is
(36,59)
unaffected by probe modification of Researchers have focused on
the fiber active tension result as proof that the system stays essentially intact
and have reasoned that the altered ATPase activities reflect modifications in
enzymatic steps of activated cross-bridges that are not usually rate-limiting
steps in muscle contraction. (59)
6.3. Discussion
Our intent in this chapter is to introduce a systematic description of N,

the angular probability density of molecular elements in a biological assembly.
The expansion in terms of order parameters in Eq. (6.1) is a description of N
that is applicable to steady-state fluorescence polarization (Section 6.2.2),
time-resolved fluorescence depolarization (Section 6.2.3), and electron spin
resonance (Section 6.2.4). We find that we can distinguish experimental
techniques based on their angular resolution. This property proved to be use-
ful in our investigation of cross-bridge orientation during muscle contraction.
One important avenue for classical optical research, which could have
immediate application in biophysics, is the investigation of experimental
methods to increase the angular resolution of fluorescence polarization techni-
ques. We are aware of two interesting possibilities, which we describe briefly
below.
The first method comes from the application of total internal reflection
fluorescence polarization techniques to study the probe spatial and angular
probability densities in two-dimensional layers.(60–69) This work brought to
light several useful properties of fluorescent light emission from probes near
dielectric interfaces. (9,70–74) When a fluorescent probe is near a dielectric inter-
face (within a distance of approximately a wavelength of the interface), we
showed that the interface can perturb the near field of the dipole and cause
the observed electromagnetic emission pattern of the fluorophore to be greatly
altered from the typical dipolar emission pattern. (9) Axelrod and co-workers
have since pointed out that the rate of total energy dissipated by a fixed-
amplitude dipole varies with its orientation and distance from the interface
and the nature of the interface coating. (75,76) They showed that a power-
normalized dipole is the correct model of a fluorophore under constant
illumination. We derived the power-normalized fluorescence polarization

signal from fluorophores near an interface and its dependence on the order
parameters and showed that the fluorescence polarization signal depends
on an infinite number of parameters such that j = 0, 1, 2, 3, ....(77) This
finding proves formally that we can increase the angular resolution of fluo-
rescence polarization measurements by observing the emission of a fluorophore
near an interface. We developed this idea and derived an analytical, model-
independent method of obtaining this information from the fluorescence
signal. Experiments are under way to take advantage of this higher resolution
technique in the study of protein components in muscle fibers.
The second method comes from the suspected observation of electric
quadrupole radiation from fluorescent probes near dielectric interfaces. This
observation raises the possibility of measuring polarized fluorescent emission
from the electric quadrupole transition. (61) The fluorescence polarization
signal from such a transition should be sensitive to order parameters of rank
greater than 4. The difficulty associated with such an approach is the small
intensity of this second-order electronic transition compared to that of a
dipole transition. It is estimated for atomic electron transitions that the inten-
sity of the electric quadrupole transition is a factor of weaker than
that of the electric dipole transition, where is the effective nuclear charge.
The value of is 1 for transitions of valence electrons, and the
atomic number, for X-ray transitions.(78) It may prove to be experimentally
infeasible to isolate the quadrupolar from the dipolar emissions for the
molecular transitions giving rise to visible or near-visible fluorescence;
however, some theoretical work is now under way in our laboratory to further
investigate this idea.
The development of higher resolution fluorescence polarization methods
is an important research objective in biophysics. Presently the fluorescence
detection of on the order of 10 fluorescent probes in the visible region is
possible. This sensitivity allows good time resolution in the observation of
dynamical processes such that picosecond spectroscopy is not uncommon. (79,80)
Presently, however, angular resolution is severely limited as pointed out in
Section 6.2.1. The combination of good time resolution and high angular
resolution in a fluorescence technique would obviously be of benefit to muscle
research and in other areas of biophysics.
Acknowledgment
The writing of this chapter was supported by a grant from the Mayo
Foundation.
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7
Fluorescence- Based
Fiber-Optic Sensors
Richard B. Thompson
7.1. Introduction
Fiber-optic sensors represent an emerging technology that will have

impact in fields as diverse as medical diagnostics, pollution monitoring,
aeronautical engineering, oceanography, and navigation. Fiber-optic sensors
operating on a variety of principles, and detecting a great variety of analytes
and influences such as temperature or pressure, have been described in the
literature (1,2) . An important subset of fiber-optic sensors utilizes fluorescence:
that is, the signal is a change in fluorescence caused by the influence or
analyte. For our purposes, “fluorescence” will serve as a generic term for
photoluminescence of all types (e.g., fluorescence, phosphorescence, delayed
fluorescence) except where distinctions are important. This chapter will con-
sider such sensors and some technical issues of their design and construction.
Since good reviews exist, (3–5) the prior art will not be comprehensively
reviewed.
The advantages of fiber-optic sensors for many applications are worth
summarizing; we are concerned both with “real-world applications” and with
the use of fiber sensors in laboratory situations. Perhaps best known is their
capability for remote, continuous monitoring of analytes or influences in real
time. A central instrument (perhaps in a benign enclosure) can monitor an
array of fibers, which may be in hostile environments. Several early applica-
tions have made use of the dielectric fiber’s immunity to powerful electro-
magnetic fields and its lack of electrical current in flammable environments.
Optical signals have an enormous inherent bandwidth (and thus information
capacity), and fibers offer a multiplex advantage since signals of different
Richard B. Thompson • Department of Biological Chemistry, University of Maryland School

of Medicine, Baltimore, Maryland 21201. This work was performed while the author was with the
Bio/Molecular Engineering Branch, Naval Research Laboratory, Washington, D.C. 20375.
Topics in Fluorescence Spectroscopy, Volume 2; Principles, edited by Joseph R. Lakowicz. Plenum
345
346 Richard B. Thompson
colors can propagate simultaneously in either direction. Fiber optics permit

optical signals to pass through tortuous passages, out of sealed chambers,
or through reciprocating machinery; evidently, a fiber itself has none of the
alignment problems of a system built using discrete optical components.
Indeed, there are some tasks which can only be accomplished using fibers.
Fiber optics can be very small and light, an important consideration in ships,
airplanes, and spacecraft.
7.2. Fiber-Optic Fundamentals
From an optical standpoint, fibers are a subclass of optical waveguides

which operate using the principle of total internal reflection. Light incident on
the interface between two dielectric media of different refractive indices will be
either reflected or refracted according to Snell’s law (see Figure 7.1). Light is
thus confined within a transparent cylinder (or fiber) if the light is launched
into it at a shallow enough angle and the cylinder is surrounded by a medium
of lower refractive index. Thus, optical fibers consist of a core of high
refractive index, surrounded by cladding of slightly lower refractive index, the
whole usually protected in a nonoptical jacket (see Figure 7.2). Fibers can be
made of fused silica, optical glasses, various plastics, even sapphire, depending
upon the desired optical properties.
Snell’s law serves to describe the macroscopic optical properties of
waveguides, but breaks down on a microscale. In particular, waveguides of
submillimeter dimension are much better described by theory that considers
Fluorescence-Based Fiber-Optic Sensors 347
their ability to propagate particular modes of light; that is, the optical power
in the waveguide will have particular spatial, temporal, and polarization
properties, which are determined by the geometry of the waveguide in a
fashion analogous to modes in a laser cavity or microwave waveguide. For
a few ideal cases, the mode distribution can be calculated exactly,(6) but in
many cases the modes a fiber will propagate are determined empirically. The
reader is referred to recent texts(6–8) for details, but some important concepts
are described below.
Some fibers are constructed with thin cores and a very small
difference in refractive index between the core and cladding, so as to conduct
only a single mode (the mode). These singlemode fibers have a number
of characteristics potentially important in fluorescence sensors. They have the
lowest possible temporal dispersion; that is, pulse spreading is minimized,
increasing the effective bandwidth (useful for telecommunications) and maxi-
mizing the resolution of time-resolved techniques (see below). Because they
are singlemode, such fibers are insensitive to influences such as temperature
or bending, which change modal distribution, and therefore propagated
intensity. Some fiber-optic properties, such as polarization preservation, are
only available in singlemode fibers.
By comparison, fibers with larger cores and/or larger refractive index
differences are termed multimode since they will propagate several modes at
once. The physical properties which determine how many modes a fiber will
accommodate are expressed in the waveguide parameter, or V-number:
where is the wavelength, is the core diameter, and and are the core
and cladding indices, respectively; a V-number of 2.405 or less ensures that
the fiber is singlemode. The waveguide parameter is wavelength-dependent,
and thus a fiber that is singlemode in the infrared will typically carry a few
modes at visible wavelengths. Among the advantages of multimode fibers is
the ease of coupling light into the fiber, since they have both larger cores and
(because of the larger difference in refractive indices) higher numerical aper-
tures (see Figure 7.2). While singlemode fibers have both core and cladding
made of glass or fused silica, plastic-clad fused-silica (multimode) fibers are
available; the plastic cladding is easy to remove, permitting access to the
core (see below). Multimode fibers with large cores are also better suited to
propagating high-powered laser (excitation) beams; a multiwatt argon laser
beam might create some interesting but unproductive effects if focused to a
micron-sized spot on the end of a singlemode fiber.
Another important phenomenon not described by Snell’s law is the
evanescent wave. When total internal reflection takes place at an interface, the
electric field strength of the incident light beam does not drop abruptly to
zero at the interface; rather, it decreases exponentially from the interface into
the lower refractive index medium over a distance of a few hundred
nanometers. The electric field present in the lower refractive index medium
can be absorbed by fluorophores near the interface and thus permits surface-
specific fluorescence spectroscopy. This phenomenon has been extensively
exploited by Harrick (9, 10) and Axelrod and Burghardt.(11, 12) Others (13, 14) have
made use of the surface specificity to devise immunoassays. As a first
approximation for multimode waveguides, the depth of penetration of the
excitation into the surrounding medium (if the cladding has been removed) is
a function of wavelength, angle of incidence and refractive index ratio of the
media (where ):
The polarization of the incident light also is a factor, and for waveguides
propagating only a few modes, the above equation is inexact. As a rule
of thumb, however, the fewer modes a fiber optic carries, the greater the
proportion of power propagated in the cladding. The evanescent wave is
important because it represents an alternate means of coupling power out of
the fiber to excite fluorescence, in addition to the distal tip.
From the standpoint of performing fluorescence experiments, perhaps the
most important property of the fiber is its attenuation at the wavelengths of
interest. Remember that in a fiber optic, one is essentially looking through a
window of glass several meters thick; the achievement of ultralow-loss fiber
optics is all the more impressive considering that exceptionally clear water is
required if we wish to see ten meters through it. Attenuation is expressed in

decibels (dB), which are defined in terms of the intensities I:
Thus, a length of fiber with 40 dB of attenuation would decrease the light

intensity at the distal end ten thousand-fold, or, in more familiar terms, have
an optical density of 4.0. Attenuation is wavelength- and fiber-dependent
(see Figure 7.3) to an extent that has a large impact on any fluorescence
experiment. For instance, a fluorophore that can be excited at 500 nm would
receive one hundred million-fold (80 dB) more light at the end of a kilometer
of the fiber profiled in Figure 7.3 than one excited with equal incident inten-
sity at 300 nm. This is one reason why most long-haul telecommunication is
performed at near-infrared wavelengths. Note that the main source of the
attenuation in the visible and near UV is Rayleigh scattering, with its
dependence, and thus most fibers exhibit similar attenuation profile shapes
at these wavelengths. Obviously, if the application requires a shorter fiber,
these considerations become less important. Conversely, a remote sensing
application might preclude the use of a fluorophore with an absorption
maximum of less than 600 nm.
7.3. Sensor Design
A large number of fiber-optic sensors employing fluorescence have been

described in the literature; we shall consider those of particular didactic value.
The reasons for using fluorescence (as opposed to other observables) in fiber-
optic sensors are well known: fluorescence is intrinsically more sensitive than
absorbance due to the Stokes’ shift and is more flexible in that a great variety
of analytes and influences are known to change the emission of particular
fluorophores. The operating principles (observables) used in several sensors
will be discussed, followed by design considerations of sensing tips, fibers
themselves, optical components of the system, and light sources.
The essence of fluorescence analysis is to understand and exploit changes
in fluorescence. These changes may be in the intensity, lifetime, color,
and/or polarization of the emission. Indeed, any interpretable observation
of fluorescence might be made the basis of a sensor. Thus, temperature
sensors available commercially are based on changes in relative intensity of
emission lines of europium: gadolinium oxysulfide or changes in lifetime of a
phosphor.(15) The former illustrates an important point, namely, that simply
measuring changes in intensity is unsatisfactory for many applications. This is
because interfering factors such as microbending, temperature, source fluctua-
tion, fluorophore degradation, and detector aging affect accuracy. Moreover,
recalibrating a sensor that may be in a remote or hostile environment could
prove difficult. Thus, several pioneering sensors that measured changes in
fluorescence intensity due to oxygen quenching, (16) binding of metal ions, (17)
or pH changes(18) have yet to come into wide use. It is unfortunate that many
of the analytical applications of fluorescence(19–21) are based strictly on
intensity changes and thus are less useful than they might be. By comparison,
spectral shifts can be measured ratiometrically, thus minimizing some of
the above problems. Use of external standards or monitoring scattered
excitation in a “double-beam” arrangement can also improve accuracy.(22, 23)
Fluorescence lifetimes(15, 24, 25) are also largely intensity-independent and there-
fore are increasingly used.
7.4. Sensing Tip Configurations
Fiber-optic fluorescence sensors have generally had only two types of

configuration of the “business end” of the sensor: the “distal cuvette” con-
figuration (Figure 7.4) or the “waveguide binding” configuration (Figure 7.5).
The distal cuvette configuration looks simple, but is seldom trivial to
construct. The fluorescent material is generally immobilized in front of the
fiber(s) within some transparent matrix that permits access of the influence
(temperature, pressure) or analyte to the fluorophore. Due to different
requirements a variety of matrices have been employed, including controlled
pore glass,(18) cellulose,(17) polyacrylamide,(26) polystyrene resins,(22) sili-
cones,(27) and liquid encapsulated in dialysis tubing. (28) The difficulties lie in
that few matrix materials are free from interfering fluorescence, permit
immobilization of the fluorophore without disturbing its properties, are
mechanically strong and durable under the experimental conditions, permit

rapid diffusion of the analyte, and can be bonded somehow to the fiber tip.
This last issue looms large when the tiny dimensions of the fiber tip (usually
<0.5 mm) are considered. Indeed, the tiny cross-sectional area of the fiber
core imposes a limit on the area (or volume) which may be illuminated;
Wyatt et al.(29) have also pointed out the photobleaching problems which may
be encountered if the fluorophore is too close to the end of the fiber. The use
of polymers which permit the fluorophore to be covalently bound in a matrix
which is itself covalently bound to the glass of the fiber will find wide use,(26)
and clearly many of the techniques developed for affinity chromatography can
be adapted for biosensing applications.(5)
The waveguide binding configuration makes use of the evanescent
wave propagating near the surface of the waveguide, as mentioned above;
essentially, surface binding (or desorption) of an analyte is detected by
changes in fluorescence intensity as it enters or leaves the zone near the
surface (see Figure 7.5). This scheme has been the basis of several
immunoassays, (l3,14,30,3l) exhibiting high sensitivity. (3) Issues that may have
hindered wide use include speed, the difficulty of immobilizing antibodies
while retaining activity and preventing nonspecific adsorption, and the sen-
sitivity attainable in a fluorimeter where only a small fraction of the exciting
light reaches the fluorophores. (5)
Indeed, the problem of coupling the light out of a fiber to excite the
fluorophore and coupling the fluorescence back into the fiber (or another
fiber) seems to be central, and yet little explored. For instance, it is easy to
show that most of the optical power in a step index singlemode fiber actually
propagates in the cladding. As we consider fibers that propagate a few, then
more modes, an ever-decreasing proportion of the power propagates in the
cladding (see Figure 7.6). Recall that for a fiber that guides a single mode at
visible wavelengths, the core diameter is less than ten microns, and the
difference in refractive indices between core and cladding is very small, less
than 0.01, yielding a V-number of less than 2.405. The fibers (rods) that are
often used in waveguide binding configurations have diameters of 600 or
greater, and the refractive index difference between them and the solution that
bathes them (and serves as cladding) is perhaps 0.17, giving a V-number of
about 4400 (Eq. 7.1). Thus, these fibers propagate a great many modes, and
only a tiny fraction of the optical power that they guide leaks out into the
medium and is available to excite fluorescence (Figure 7.6). (32) Under these
circumstances, the importance of launching the light near the critical angle
has been emphasized.(33)
There are some data that suggest that, for fluorophores near to the
waveguide surface (or fiber core), fluorescence is coupled back into the fiber
more efficiently than would be anticipated from a strictly ray optics view-
point. (34,35) If it were possible, a systematic study that examined the efficiency
of fluorescence being coupled back in as a function of refractive indices,

fluorophore proximity and orientation, and mode structure would be most
welcome. It may be that the fluorescence will be coupled back into the fiber
to the extent that the fluorescence distribution mimics the distribution of light
in the mode(s) guided by the fiber.
7.5. Fiber Characteristics
The characteristics of the fiber itself play an important role in the

design. The variety and quality of fibers made available due to their use
for communications and other applications has been an enormous benefit.
Thus, single- and multimode fibers are available cheaply, and with very low
attenuation. We have discussed the importance of fiber attenuation above.
The background fluorescence of the fiber is also a consideration.(36) A repre-

sentative spectrum is shown in Figure 7.7 for a common multimode fiber, with
excitation in the ultraviolet. The emission bands may be due to plasma lines
from the laser discharge, fluorescing metal ion impurities in the fiber, color
centers, and/or Raman scattering.(37) Evidently, as the fiber lengthens the
relative intensity of these bands will increase, lowering sensitivity if the signal
fluorescence overlaps the background bands. A several authors have pointed
out, time-resolved methods offer the prospect of avoiding this background, as
illustrated in Figure 7.8. Essentially, one sends a brief pulse of exciting light
down the fiber and waits to turn on the detector until the fluorescence is
expected to return; photoluminescence originating elsewhere within the fiber
will arrive at the detector at different times depending on its intrinsic life-
time and position and will not be detected. Note that this is one of the few
instances where phase methods are likely to be inferior to time-resolved
methods, since in order to utilize phase-sensitive detection methods( 38,39)
successfully, the photon transit time (fiber length) should be a harmonic of the
lifetime.
A second fiber-optic characteristic, called dispersion, limits the fiber
bandwidth and thus can affect time-resolved fluorescence measurements
through fibers. Dispersion is of two types, termed modal dispersion and
material dispersion. Modal dispersion occurs in multimode fibers and arises

from differences in the group velocities of various modes propagating in the
waveguide; a detailed treatment is available. (8) More intuitively, rays launched
into the fiber near the critical angle travel a longer path getting to the distal
end than those launched parallel to the fiber axis, and thus a short pulse of
light will be broadened by the time it reaches the end of the fiber. This
property is usually expressed as a frequency–distance product: that is, a fiber
specified as having a 300-MHz-km product would carry a 300-MHz signal
1 km with less than 3-dB loss due to demodulation. Note that graded index
fibers are specifically constructed to minimize modal dispersion. (6, 8) By their
very nature, singlemode fibers do not suffer from modal dispersion.
Material dispersion affects all fibers and is a limiting factor in singlemode
fiber bandwidth. It arises from the variation of refractive index (light velocity)
with wavelength of the glass used in the fiber. A red photon travels faster
through most fibers than a blue one, and thus a pulse of monochromatic light
will be less broadened (temporally) than an equally short one having a larger
(wavelength) bandpass (Figure 7.9). High data rate communications applica-
tions therefore require sources which can be rapidly modulated and also are
very monochromatic. Since material dispersion is a property of the glass, the
fiber may be constructed to minimize the material dispersion in a particular
wavelength regime, usually one of the low-attenuation (IR) windows. Finally,
carefully controlled material dispersion is important in fibers used in “pulse
compressors.”(40)
7.6. Separating Excitation and Emission
How exciting light is coupled into and fluorescence collected from the
fiber is also an important design question. The simplest solution is to employ
different fibers for guiding excitation to and fluorescence from the sensing tip.
This has the advantage of simplicity in design and permits optimizing the fiber
for each wavelength. However, it also has drawbacks: the receiving fiber easily
collects scattered excitation, it precludes the use of the waveguide binding
configuration, and it multiplies the engineering problems of the distal cuvette
configuration. These last include the difficulty of registration: exciting the
same volume of sample that the emission fiber is capable of viewing, and
attaching and sealing the sensing tip. Thus, this geometry is less favored by
current workers in the field.
By contrast, use of a single fiber to guide excitation and emission
simultaneously avoids these problems and makes use of the fiber’s natural
advantages. In this case, it is necessary to separate the excitation and emission
based on their differing wavelengths or spatial characteristics; a number of
configurations have been described for doing this (Figure 7.10A, B). The two
most common make use of multilayer dielectric-coated mirrors or interference
filters. In Figure 7.10A the interference filter (with the depicted spectrum)
passes the exciting light, while reflecting the emission; the dielectric-coated
mirror in Figure 7.10B does the converse. The configuration in Figure 7.10A
suffers some loss because the interference filter (which may be angle-tuned to
admit the desired wavelength) seldom passes more than 30% of the light at
the peak. The configuration in Figure 7.10B takes advantage of the fact that
mirrors with multilayer dielectric coatings can be outstanding reflectors, with
coatings available that reflect 99 + % of particular laser lines, while passing
longer wavelengths. Problems can arise with this configuration, however,
when powerful (laser) or short-wavelength sources are used. In both con-
figurations the excitation hits the filter directly and often causes it to fluoresce
itself. The importance of this problem varies with the particular filter, the
wavelength of the signal fluorescence, and the sensitivity required. It is of course
generally true that the greater the sensitivity required, the more important
obscure sources of background fluorescence become. The configurations in
Figure 7.10C (3) and D use a spatial filtering method for separating (laser)
excitation from emission coming back out of the fiber. In both cases the
highly collimated laser beam passes through the hole in the mirror into the
fiber; the fluorescence comes out with a conical distribution determined by
the fiber’s numerical aperture and is reflected by the mirror into the detector.
The drawback to this method is that such mirrors must often be custom-made.
The diameter of the laser beam, the numerical aperture of the fiber, and the
magnification of the coupling lens all affect the placement of the mirror and
the size of the hole. Thus, a fiber with a low numerical aperture would need
the mirror placed far from the coupling lens to maximize the fluorescence
collected. The configuration in Figure 7.10D is simply an extension of that in
Figure 7.10C which eliminates the collecting lens; we have found that a small
aspheric mirror produced by Polaroid(41) is satisfactory for this purpose.
7.7. Launching Optics
Up to this point, we have neglected the lens(es) used to couple light into
and out of the fiber. It is fortunate that microscope objectives often fulfill most
of the desiderata for such lenses, being highly corrected, compact, widely
available with a variety of characteristics, and relatively inexpensive, con-
sidering their quality. Design, construction, and alignment of comparable
optical systems using discrete lenses would be slow and costly. In fact,
microscope objectives of low power are well suited to coupling light
into multi- or singlemode fibers. Practical concerns here include matching the
angle of launching and spot size to the fiber numerical aperture and core size
or matching mode distributions in singlemode fibers.(7) Most microscope
objectives have low loss in the visible region of the spectrum, but transmission
falls off abruptly in the near UV; also, the glass in the objective can fluoresce
as well. Quartz objectives that minimize these problems are available, but are
very expensive. Reflective objectives are also expensive and are tricky to use
with lasers, but are largely wavelength-independent (see Figure 7.7). Other
optics used for coupling light into fibers include spherical lenses and gradient-
index (GRIN) rods,(7) which are akin to gradient-index fibers. These small
devices are best suited for use with laser sources and are less convenient to use
than microscope objectives; they are widely used to couple diode laser output
into fibers.
7.8. Light Sources
A great variety of light sources have been used with fluorescence-based

fiber-optic sensors. Incoherent sources such as lamps or light-emitting diodes
have been widely used until now because they are inexpensive, offer wavelength
variability, can be very rugged, and are simple and reliable. Despite these
attributes, for many purposes lasers are better sources, mainly because they
are brighter and easier to couple into fibers. Readers unfamiliar with laser
characteristics are referred to texts (42) and catalogs(43) for more information.
An important advantage of lasers for fluorescence sensors is their narrow
bandwidth compared with that of lamps; the lack of scattered stray light
at the fluorescence wavelength eliminates a common source of background.
Many visible lasers operate at a single line, and multiline lasers can be easily
tuned. Most gas lasers also produce plasma lines at wavelengths differing from
the laser wavelength, which may mimic the fluorescence to be detected
(Figure 7.7). These plasma lines are incoherent and uncollimated, however,
and can be eliminated by a monochromator or spatial filter; the perforated
mirrors in Figure 7.10C and D are well suited for this.
Several attributes of a laser must be taken into account for fluorescence
sensors. Foremost is the wavelength of the laser, which is constrained by (or
constrains) the choice of fluorophores. In particular, a probe having a lower
peak extinction coefficient or quantum yield might still provide more signal,
if it could be excited more effectively by a particular laser line. (5,44) There
are several lasers available with output at wavelengths above 500 nm, but
comparatively few with output in the blue and UV regions. The paucity of
UV laser types, along with the high attenuation of fibers in the UV (see
Figure 7.3), is unfortunate, given that many interesting and useful fluorescent
probes are excited in this wavelength range. Future probe development will
likely be at wavelengths ranging into the near IR, where attenuation is low,
bandwidths are high, and a variety of sources are available.
In addition to wavelength, other attributes of the laser are important: its
power, stability, spatial and temporal characteristics, cost, and reliability are
all factors in the laboratory. For laser-based sensors employed in the field or
harsh environments, the cooling, space, and electrical power requirements may
also be important. Output power must be great enough to provide adequate
sensitivity, yet not so great as to damage components of the system or create
nonlinear effects. Damage is wavelength-dependent: picosecond microjoule
pulses (having very high peak powers) in the infrared may be compressed
in singlemode optical fibers,(40) but coupling even modest output from an
excimer laser in the far UV into a large-core silica fiber remains difficult.
Stability, both in intensity and beam pointing, is a concern, but most lasers
are adequate in this respect. The temporal characteristics are primarily of
interest for time-resolved experiments, and they are dealt with in the next
section.
The recent trend toward development of low-power solid-state lasers
promises to have a large impact on fluorescence-based fiber-optic sensors. The
diode-pumped YAG lasers with output frequency-doubled in the green† are
indicative of this trend, being all solid state, small, lightweight, and rugged,
and having low power consumption. At present, they remain expensive and of
modest (< 5 mW) output, but both characteristics are certain to improve.
Prototype continuous-wave (cw) diode lasers emitting frequency-doubled
(blue) output have been shown.(45) The commercial availability of gas lasers
†
Diode-pumped YAG lasers with frequency-doubled output in the green are currently available
from Spectra-Physics, Moutain View, California; Amoco Laser, Inc., Naperville, Illinois, and
AB Lasers, Concord, Massachusetts.
emitting lines heretofore only demonstrated in laboratory prototypes is very

welcome: the recent availability of argon output in the 275–305-nm band is of
particular interest to biophysicists. Similarly, helium–neon lasers emitting at
543 nm are notable because they offer a wavelength useful for exciting
fluorescence in a package that is rugged, convenient, and potentially battery
powered. The milliwatt output of most of these lasers is hardly a drawback,
given the sensitivity of fluorescence, and their typically low power consump-
tion frees the practitioner from the tyranny of water cooling.
7.9. Time-Resolved Fluorescence in Fibers
The utility of temporal resolution in fluorescence studies of all kinds is

practically self-evident, particularly to readers of these volumes. Two questions
arise: can one do the same sort of time-resolved experiments “through” fiber
optics, and what are the problems in using fibers for such experiments? In fact,
time resolution is already employed in commercial fiber-optic temperature
sensors(15) and offers the prospect of substantial background reduction
(Figure 7.8). In addition to the well-known pitfalls of frequency- and time-
domain measurements,(46,47) fiber dispersion (discussed above) can pose
problems. Either modal or material dispersion can impose a limit on the
time resolution of a fluorescence measurement, or on how far away it can be
made. A typical value of modal dispersion in a multimode fiber might be
300 MHz-km, making subnanosecond measurements through kilometers of
fiber difficult; however, use of a gradient-index fiber could reduce this substan-
tially. A typical value for material dispersion in a singlemode waveguide at
633 nm might be 300 ps/nm per kilometer. Thus, a short pulse from a synch-
pumped dye laser having a linewidth of 0.01 nm would be lengthened by only
a few picoseconds on going through a kilometer of fiber, but fluorescence
emission over a 50-nm band passing back through that kilometer of fiber
would experience dispersion over 15 ns, which would have to be deconvoluted
from the fluorescence lifetime itself. An expensive solution to this is to
design and construct the fiber to have zero (or negative) material dispersion
in the wavelength range of interest. Note that the material dispersion is wave-
length- and material-dependent, (8) typically decreasing going from the visible
to the infrared and becoming zero at or so. From the standpoint of
fluorescence measurements, this effect could be the source of artifacts similar
to the timing errors introduced by the color effect in photomultiplier tubes.
An additional factor is the availability of light sources. The sort of pulsed
lamp source which is convenient for time-correlated single-photon counting is
difficult to use with fibers because of its low intensity, poor collimation, and
large working distance. Pulsed laser sources are better choices for fiber
applications for the reasons described above. In addition to the well-known
problems involved in using pulsed or cw mode-locked lasers for time-resolved

measurements,(47) there are additional caveats. In particular, if the spectral
linewidth of the laser is broad, the pulses will be temporally broadened due
to the material dispersion. Also, short pulses having very high peak power can
experience nonlinear effects in the fiber due to its long interaction length.
Frequency-domain measurements would appear to be technically easier to
carry out than time-domain measurements, given that the intensity modulation
of (more widely available) cw laser, lamp, and diode sources is straight-
forward.(24) To date, relatively few reports of time-resolved measurements
through fibers have appeared in the literature; advances in technology and the
obvious utility of the technique both suggest that it will play an important
role.
7.10. Polarization
While fluorescence polarization remains an important tool in biochemical

studies,(48) it has been little employed using fibers. Ordinary fibers become
birefringent when stressed by bending, causing fluctuations in the propagation
of polarized modes. “Polarization-preserving” singlemode fibers propagate
orthogonally polarized modes at different speeds. Thus, linearly polarized
light launched into the fiber at some angle to its axes of stress will be in turn
linearly, circularly, and elliptically polarized as it passes down the fiber and
the relative phases of the E-vectors change.(7) Using coherent sources, if some
mode coupling between the orthogonally polarized modes in the polarization-
preserving fiber occurs as a result of microbending or just long interaction
length, then interference and intensity fluctuations occur. Also, coupling
uncollimated fluorescence into singlemode fibers may prove difficult. Despite
these concerns, the utility and ratiometric nature of fluorescence polarization
measurements argue that fiber-optic sensors will ultimately be designed to use
them.†
7.11. Conclusion
We have discussed some of the special problems and opportunities that

result from employing fiber optics in fluorescence sensors and experiments. It
should be evident that fiber optics do not represent a panacea, but rather a
means for solving particular problems—sometimes elegantly. One purpose of
this chapter was to stimulate the community to apply this technology to new
†
Companies offering polarization-preserving fibers include Andrew Corporation, Orland Park,
Illinois, and York V.S.O.P., Princeton, New Jersey.
experiments in biochemistry and biophysics; from the experimental

demonstration of such techniques come the practical applications of
tomorrow.
Acknowledgments
I would like to thank Susan McBee and Lynne Kondracki for their help
in preparing the figures; Frances Ligler, David Kidwell, and Carl Villarruel
for helpful discussions and careful reading of the manuscript; Michael P. Eden
of Polaroid Corporation for the gift of an aspheric mirror; and the Office of
Naval Technology for support.
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8
Inhomogeneous Broadening of
Electronic Spectra of
Dye Molecules in Solutions
Nicolai A. Nemkovich, Anatolyi N. Rubinov, and
Vladimir I. Tomin
8.1. Introduction
It is well known in spectroscopy of complex organic molecules that the

large width of their absorption and emission bands is largely due to the
existence of a continuous set of vibrational sublevels in each electronic state.
The spectroscopic properties of dye molecules in solution are in addition
influenced by the surrounding medium. (1–3) For several decades it had been
believed that because of the fast energy exchange between the vibrational sub-
levels the fluorescence spectrum of organic dye in solution was independent of
the frequency of the exciting light. In 1970 it was shown (4) for the first time
that apart from molecular vibrations there is another cause of the substantial
broadening of electronic spectra of organic molecules in solution, namely, the
fluctuations of the structure of the solvation shell surrounding the molecule.
The variation of the local electric field caused by the fluctuation of the shell
structure leads to a statistical distribution of the frequencies of the electronic
transitions of the molecules and, therefore, to inhomogeneous broadening of
the dye spectrum.
This broadening was experimentally demonstrated (4–6) from the
dependence of the fluorescence spectrum of a dye solution on the exciting
radiation frequency at 77°K. Later, Personov et al.(7) observed inhomo-
geneous broadening of electronic spectra from frozen solutions of complex
molecules at lower (liquid helium) temperatures. It was shown that at liquid
helium temperatures discrete fluorescence spectra of complex molecules with
Nicolai A. Nemkovich, Anatolyi N. Rubinov, and Vladimir I. Tomin • Institute of Physics of

the B.S.S.R. Academy of Sciences, Minsk 220602, U.S.S.R.
367
368 Nicolai A. Nemkovich et al.
resolved vibrational structure could be obtained by eliminating the inhomo-

geneous broadening by means of selective monochromatic excitation of the
solution. This made it possible to gain a deeper insight into the nature of the
electronic spectra of complex molecules and led to new, more sophisticated
methods for their study. It has been shown (8,9) that exposure of a frozen
solution of organic molecules at liquid helium temperatures to sufficiently
intense monochromatic radiation results in a selective energy gap in their
inhomogeneously broadened absorption spectrum. Such an energy gap may
exist for a long time, which opens up new possibilities of studying very subtle
spectroscopic effects in complex molecules and of using such systems as
optical memories. Subsequently, it was shown (10–12) that inhomogeneous
broadening of electronic spectra of complex molecules occurs not only in solid
solutions but in liquid solutions, too. In the latter case, it is dynamic in nature
and can only be detected by nano- or picosecond time-resolved spectroscopic
techniques.
The systematic study of inhomogeneous broadening offered an essentially
new picture of the nature of the spectra of complex molecules in solutions at
various temperatures and revealed new spectroscopic effects and features.
Thus, the “up-relaxation” effect was discovered.(10) This effect manifested itself
as an increase in the energy emitted by a fluorescent molecule during con-
figurational relaxation of the solvation shell surrounding it. In other work, (13)
the phenomenon of directional nonradiative energy transfer from the “blue” to
the “red” centers of an inhomogeneous ensemble of complex molecules in
solid solutions was experimentally observed. This phenomenon explains such
well-known effects as the Weber effect (14–17) and the red shift of fluorescence
spectra with increasing concentration in such systems,(18) and it gives a deeper
insight into the mechanism of energy transport in the photosynthetic system
of plants.(19, 20)
Finally, optically induced dye molecule rotation due to the configurational
relaxation of nonequilibrium solvates was observed in polar solutions. (21) The
effect depends on the excess of configurational energy of selectively excited
solvates and leads to specific dependencies of the kinetics of radiation
anisotropy on the exciting light frequency and the frequency at which the
emission is recorded.
Thus, the discovery of inhomogeneous broadening of electronic spectra of
complex molecules in solid and liquid solutions gave rise to a new field,
namely, the selective spectroscopy of organic solutions. The main feature of
this field is the selective excitation of some members of an inhomogeneous
ensemble of molecules, which enables them to be studied and treated selectively.
This chapter is not intended as a comprehensive review of studies of
inhomogeneous broadening. We will just consider the principal physical
features of this phenomenon and the main spectroscopic effects associated
with it.
Inhomogeneous Broadening of Electronic Spectra of Dye Molecules 369
8.2. Theoretical Considerations of Inhomogeneous Broadening
8.2.1. Solvate Configurational Energy
The spectroscopic properties of a dye solution may be analyzed by treating

its elementary cell (solvate), which includes a fluorescent organic molecule
and its immediate surroundings. Due to the statistical variation of the cell
structure, different cells may have different local electric fields. To describe this
situation phenomenologically, a solvate field diagram (representing the poten-
tial energy of the solvate versus the reactive field strength) was introduced.(22)
The use of such a diagram makes it easier to analyze the inhomogeneous
broadening of electronic spectra of dye molecules in solution in order to
understand its various spectroscopic manifestations. In this section we analyze
the inhomogeneous broadening characteristics of fluorescent organic molecules
in solid and liquid solutions using a classical solvate model suggested in
Ref. 12.
Without specifying the dimensions and spatial configuration of the
solvation shell, we will treat it in terms of its macroscopic characteristics, that
is, its susceptibility, just as various dielectric materials are treated.
First, consider a polar solution in which the solute molecules possess
constant dipole moments. In each elementary cell of such a solution the
immediate surroundings are polarized due to the dipole moment, of the
dye molecule, thus giving rise to a reactive field, in the cell:
where is the susceptibility of the solution.

Inhomogeneous broadening occurs because, as a result of the thermal
motion of molecules in a solution, different cells have different solvation shells
and, therefore, different reactive fields R, which differ somewhat from the
mean value of found in Eq. (8.1). As the 0–0 transition frequency of
the dye molecule depends on the reactive field intensity, this means that in the
solution there is an inhomogeneous set of molecules specifically distributed
in frequencies of the 0–0 transition. This distribution is the fundamental
characteristic of inhomogeneous broadening, hereinafter referred to as the
inhomogeneous broadening function, The function can be found if
the dependence of the 0–0 transition frequency on the strength of the reactive
field R and the elementary cell distribution with respect to field intensity are
known. Since as a function of R is well known, (1) the problem is one of
finding the distribution over R.
Let us assume in the simplified model under consideration that two forces
are responsible for the elementary cell state: (1) the polarizing force which is
due to the presence of a constant dipole moment in the dye molecule and
induces the reactive field R in the cell; and (2) the restoring force which is due
to the action of the reactive field on the dipolar molecules of the solvation shell.
The action of the restoring force becomes evident if we consider what would
happen if the dye molecule were extracted from the cell. In this case the cell
would tend to reach its initial state at under the action of the restoring
force. The cell is in its equilibrium state when the two forces cancel each
other. This state is the most stable one, and it is characterized by the reactive
field Any deviation from this state requires some work to be done. The
latter will just be the measure of the potential energy of the nonequilibrium
solvate configurational interaction. Obviously, an absolute thermodynamic
equilibrium must lead to a Boltzmann equilibrium distribution of cells with
respect to the energy of the configurational interaction. Thus, the problem
is reduced to determining the configurational energy of the cell versus its
reactive field R.
Taking into account that the reactive field R is parallel to the dipole
moment inducing this field, the dipole–field interaction energy can be
represented as
This energy is essentially the work done by the dipole to reorient (polarize)
an elementary cell of the solution. The work necessary to perform such
reorientation of the solvation shell and to change the reactive field by dR can
be written in differential form as
It can easily be seen that in the solvate and R may be treated as a polarizing
force and the generalized coordinate for the solvation shell configuration,
respectively. The restoring force acts along with the polarization force
in the solvate. Thus, the total force affecting the solvation shell is
For the cell configuration corresponding to the reactive field both

forces are equal, and
Since we are considering the dipole–dipole interaction, the restoring force
must obviously be proportional to the field:
Assuming that at the total force is zero and taking into account
Eqs. (8.1) and (8.4), we obtain the proportionality factor
Consequently, the restoring force is of the form
Thus, according to Eqs. (8.1) and (8.5), the total force acting upon the
solvation shell is
It can be seen from Eq. (8.8) that the total force is zero only for cells with
Such cells are the most stable ones and possess minimum configura-
tional interaction energy.
The deviation of the cell configuration from the equilibrium configuration
involves an increase in energy as compared to the minimum value. The
magnitude of this excess may be considered as a measure of the potential or
free energy of the nonequilibrium solvate. For a cell with the reactive field R,
this energy can be determined as the work required to restructure the solvate
from its equilibrium state with the field to the state with the field R:
As can be seen from Eq. (8.9), a plot of the potential energy of the solvate
configurational interaction versus the internal reactive force has a parabolic
shape with its minimum at
8.2.2. Field Diagram of a Polar Solution
It is convenient to introduce the total solvate energy, which is the sum of

the configurational and electronic energies of the solvate molecules.† Since we
are considering electronic transitions of the dye molecule only, the electronic
energy of all the solvation shell molecules can be assumed invariable and equal
to zero. We will call elementary cells with the dye molecule in its ground
and excited states unexcited and excited solvates, respectively. Assuming the
electronic energy of the ground state of the dye molecule to be equal to zero,
the total unexcited solvate energy will be described by Eq. (8.9).
The electronic transition frequency of the dye molecule as a function of
the reactive field of the solvate can be written (1) as‡:
†
In thermodynamics it may be called “the free energy of the system.”
‡
For simplicity, we will consider the electronic transition with a constant dipole moment of the
dye molecule changing its magnitude rather than orientation. We will also neglect the quasi-
continuum of vibrational states of a complex molecule in this section.
where and are the dipole moments of the dye molecule in

the ground and excited states, respectively, and is the 0–0 transition
frequency of a free (noninteracting) molecule.
Upon absorption of a quantum hv and transition of the solvate to the
excited state, the energy of the solvate becomes, according to Eqs. (8.9) and
(8.10), equal to
This equation takes into account that the reactive field for an equilibrium
solvate in its excited state is equal to
As seen from Eq. (8.11), the potential energy of the excited solvate is repre-
sented by the same parabola as for the unexcited state, but raised by
and it has its minimum at a value of the reactive field

Thus, we have arrived at the field diagram (Figure 8.1) that was intro-
duced earlier on a purely phenomenological basis.(11) This diagram represents

the dependence of u, the sum of the orientational interaction, and electronic
energies of the molecules in the first coordination sphere, or the potential
energy E of the solvate on the electric field R. This dependence is similar to
that for the potential energy of a molecule, with the differences that, instead
of a molecule, the solvate as a whole is considered and that, instead of the
vibrational energy, the molecular configurational interaction energy in the
solvate is considered. The solvate field intensity is naturally the generalized
coordinate.
This diagram can conveniently be used to analyze the spectral properties
of a solution, and it automatically takes into account the inhomogeneous
broadening. The shell structure and, therefore, the field remain unchanged
during the electronic transition. As in the case of molecules, the Franck-
Condon principle is in force here; that is, quantum transitions between the
potential curves are given by vertical lines on the diagram. For unexcited
solvates, the equilibrium Boltzmann distribution of the configuration energy
is normally valid. The configuration energy distribution for excited solvates
depends on the relation between the lifetime of the excited fluorescent
molecule and the configurational relaxation time, of the elementary cell. At
the equilibrium distribution of excited cells over the configurational
states is attained, whereas in the reverse case the distribution function is non-
equilibrium and depends on the exciting light frequency.
In Figure 8.1a, we may recognize two characteristic transition frequencies
for the inhomogeneous ensemble of molecules under consideration: corre-
sponding to the 0–0 transition of solvates which have the equilibrium
configuration in the unexcited state, and corresponding to the 0–0
transition of solvates whose configuration is the equilibrium one in the excited
state. According to Eq. (8.10) and taking into account Eqs. (8.1) and (8.12),
these frequencies are given by
The field diagram in Figure 8.1a corresponds to the case in which the
dipole moment of the molecule increases on excitation
In this most common case, the solvent causes both inhomogeneous broaden-
ing and a general spectral shift to ward longer wavelengths (Figure 8.10)
(negative solvatochromism). When the solvent causes a spectral shift
to ward shorter wavelengths (positive solvatochromism), and the field
diagram of such a solution has the form shown in Figure 8.1b. The following
analysis of the inhomogeneous broadening characteristics holds equally for
both cases.
8.2.3. Solvate Distribution in Configurational Sublevels
If the lifetime of unexcited solvates is long enough (i.e., moderate exciting

light intensities are used), the distribution of solvates in energy of configura-
tional interaction is of the Boltzmann form:
where c is a normalization factor, and is the statistical weight of the

state with energy Substituting Eq. (8.9) into Eq. (8.16), we obtain the
solvate distribution as a function of the local field R:
where is the width of the distribution function.

Thus, the distribution of solvates with respect to fluctuations of the local
field is of a symmetric Gaussian form, and the width of the distribution is
uniquely determined by the susceptibility of the solution and the temperature.
It may be assumed that the local field fluctuations are caused by
the susceptibility fluctuations of different elementary cells of the solution.
According to Eq. (8.1),
Substitution of Eq. (8.18) into Eq. (8.17) gives the susceptibility fluctuation
distribution function for the cells
where is the width of the distribution function.

Of particular interest is the solvate distribution over the electronic
transition frequencies of the dye molecules. Using Eqs. (8.14) and (8.1), the
electronic transition frequency Eq. (8.10) can be given as
Using this expression, we obtain from Eq. (8.17)
where
Thus, the inhomogeneous broadening function characterizing the solu-

tion absorption spectrum is found to be of a symmetrical Gaussian form with

its maximum at Inhomogeneous broadening (Eq. 8.22) depends on the
difference between the dipole moments of the dye molecule in its ground and
excited states as well as on the solvent susceptibility and temperature. The
temperature dependence of exists until the freezing point of the solution
is reached. Then, the spatial configuration fluctuations of the elementary
cells become fixed, and the inhomogeneous broadening function becomes
temperature-independent. When calculating the width of the inhomogeneous
spectrum for a frozen solution, the melting point should be substituted for T
in Eq. (8.22), irrespective of the real experimental temperature.
As can be seen from Eq. (8.22), inhomogeneous broadening depends on
the susceptibility of the medium. Employing the Onsager model for a cell in
the solution, the value of can be found from the formula
where is the dielectric constant of the medium, and a is the Onsager sphere
radius. Using this expression, Eq. (8.22) may be written in the form
where
The Onsager sphere radius is governed by the size of the solute

molecules. Thus, according to Eq. (8.24), inhomogeneous broadening depends
not only on the dipole moments of the dye molecule but also, and most
essentially, on its dimensions. The smaller the molecule, the stronger is the
inhomogeneous broadening effect. This agrees with experimental data:
inhomogeneous broadening is most pronounced for small organic molecules,
for example, phthalimides.
The estimates of presented in Table 8.1 for typical polar solutions, for
example, 3-aminophthalimide in ethanol, show that, at ranges

from 400 to and that it decreases to at the freezing
point of the solution (158°K). In obtaining these estimates, the following
values were used in Eqs. (8.22) and (8.24):
8.2.4. Nonpolar Solutions
Above we have considered the case in which the fluorescent molecule

has a constant dipole moment which changes as the molecule goes to the
excited state. Inhomogeneous broadening will also manifest itself when the
luminophore molecule does not possess a constant dipole moment either in its
ground or in its excited state. The solvent may be polar or nonpolar and is
characterized by its susceptibility
In this case the equilibrium state of the cell will obviously correspond to
the local field However, because of the thermal motion of molecules in
the solution, nonequilibrium states for the cells with will occur
alongside the equilibrium ones. The configurational energy of such a cell, or
rather of its solvation shell, can be found from Eq. (8.9) assuming
The local fluctuation field R acting on the dye molecule will polarize it, thus
creating an induced dipole moment:
where is the molecular polarizability. Thus, along with the configurational

energy of the solvation shell, the nonequilibrium cell is also characterized by
the energy of interaction between the induced dipole moment and the local
fluctuation field. The absolute value of this interaction energy is
The total configurational–interaction energy of the elementary cell is the sum

of Eqs. (8.25) and (8.27):
On transition to the excited state, the molecular polarizability changes and

becomes equal to In addition, the energy of the cell increases by the energy
of the absorbed light quantum. It can easily be seen that the total energy of
the excited solvate will be
where is the electronic transition frequency of a free molecule. The solvate

field diagrams for and are given in Figures 8.2a and 8.2b,
respectively. In both cases, the parabolas lie one above the other and have
their minimum at but they differ in shape. The electronic transition
frequency for the cells with the local field is
Taking into account that unexcited cells obey the Boltzmann law and using
Eq. (8.28), we obtain the solvate distribution as a function of local field
fluctuations:
where
(8.32)
As seen, in this case also, the cell distribution over local field fluctuations is
of a Gaussian form. The distribution over electronic transition frequencies is,
however, of a different form. We obtain from Eqs. (8.30) and (8.31)
where
Thus, for nonpolar dye molecules there is an asymmetric exponential distribu-

tion of cells over electronic transition frequencies with a sharp boundary at
the frequency For molecules with the exponential rise is toward
shorter wavelengths; for molecules with toward longer wavelengths.
Estimations of by Eq. (8.34) for typical systems similar to those
investigated experimentally yield for a polar solvent with
(e.g., butyl bromide) and for a nonpolar solvent
(e.g., n-hexane). A dye molecule of the coronene or perylene type was
tested; its polarizability was estimated by the valence-optical scheme as
and and a were assumed to be and
2 Å, respectively. The estimates agree fairly well with the experimental data
obtained for the inhomogeneous broadening function by the
method of double scanning for a perylene solution in n-hexane.(23)
8.2.5. Selective Excitation with Vibrational Spectral Broadening
The spectra of a molecule in solution can be appropriately described only

with knowledge of both the inhomogeneous broadening function and the
homogeneous molecular spectra, that is, the absorption and fluorescence
spectra of molecules in cells of the same configuration. If the homogeneous
spectrum is essentially narrower than the inhomogeneous one, both the
shape and the width of the experimentally observed spectra will mainly be
determined by the inhomogeneous broadening effect and will be described by
Eqs. (8.21) and (8.31). In this case, monochromatic radiation provides
selective excitation of a given group of elementary cells of an inhomogeneous
ensemble. When the homogeneous spectrum is wider than the inhomogeneous
one, the inhomogeneous broadening effect can only weakly affect the shape of
the experimentally observed spectra, and, generally speaking, the possibility of
selective excitation of individual solvate groups is lost.
The homogeneous spectra of complex molecules at moderate temperatures
(essentially above liquid helium temperature) are defined by the presence in the
molecule of a quasi-continuum of vibrational states. As is well known,(24, 25)
energy exchange between these states occurs in extremely short times (less than
10–13 s), and this is responsible for the homogeneous nature of vibrational
broadening.
Below, a specific situation is shown to obtain for complex molecules in
solution. Selective excitation of solvates proves to be possible even when the
width of the homogeneous spectrum exceeds that of the inhomogeneous
spectrum. Thus, the inhomogeneous broadening effect will be apparent in

practically all cases.
Let the solution be excited by radiation of frequency v. As seen from
Figure 8.1, photons of this frequency will be absorbed both selectively, by
nonequilibrium solvates of type i, for which the frequency of the 0–0
transition in the dye molecule coincides with the exciting light frequency, and
nonselectively, by equilibrium solvates with the type I configuration. First,
consider the case where In this case, the radiation of frequency v will
only be absorbed by those equilibrium solvates in which the dye molecule has
excess vibrational energy equal to
To characterize the degree of excitation selectivity, we introduce the quantity

which is the ratio of the number of selectively excited solvates of type i
to the number of nonselectively excited solvates of type I (i.e., solvates which
had the equilibrium configuration in the ground state):
The values of and can be found from the balance equations if the
Boltzmann distribution of the configurational and vibrational energies of the
particles is taken into account:
Since the configurational energy distribution is continuous, n(E) is the

number of solvates per unit energy for the energy value of E.
For equilibrium solvates and excitation at a steady-state
radiation density U, the balance equation is of the following form:
where is the fluorescent lifetime of a molecule in the solvate with the type
I configuration, and is the Einstein coefficient defining the absorption of
such solvates at a frequency v. Using Eqs. (8.35) and (8.37), we obtain, from
Eq. (8.38),
where n is the total concentration of unexcited solvates.

For nonequilibrium solvates of the ith type the balance equation has the
form
and, consequently, the number of excited particles is
where is the Einstein coefficient for absorption, and is the fluorescent

lifetime of a molecule in a cell of the ith type. Substituting Eqs. (8.39) and
(8.41) into Eq. (8.36) and using Eqs. (8.9) and (8.20), we obtain for the degree
of selectivity:
Here is the width of the inhomogeneous broadening function, given for

polar molecules by Eq. (8.22). For simplicity, assume the homogeneous
spectrum to be of the Gaussian form:
where is the 0–0 transition frequency for ith type solvates, and is the
width of the homogeneous absorption band. For ith type solvates resonantly
excited by frequency v, the condition is fulfilled, and hence
For the type I solvates, and the Einstein coefficient for the exciting
light frequency is equal to
Substituting these values of and into Eq. (8.42), we obtain for the
spectral excitation range (corresponding to the long-wavelength slope of
the total absorption band) the following expression for the degree of selectivity:
A similar analysis can be made for the exciting light frequency range
(corresponding to the short-wavelength slope of the total absorption band).
The difference between these two cases is that radiation of frequency
can be absorbed by all the equilibrium solvates irrespective of the vibrational
energy stored by the dye molecule. Integration of the expression analogous
to Eq. (8.38) will therefore cover the range from 0 to For excitation
frequencies this results in
It follows from Eqs. (8.45) and (8.46) that the excitation selectivities for the
blue and the red edge of the total absorption band of a solution are essentially
different. Excitation can be regarded to be selective when since only in
this case is the number of selectively excited nonequilibrium solvates greater
than that of nonselectively excited equilibrium solvates.
Following Eq. (8.46), in the case where and is assumed
to increase with blue-edge selective excitation is only possible for
that is, under the trivial condition that the width of the inhomo-
geneous spectrum is wider than that of the homogeneous spectrum. For
complex molecules, the width of the vibrational (homogeneous) spectrum
amounts to hundreds of wave numbers; that is, it is of the same order of
magnitude as the width of the inhomogeneous spectrum. Therefore, no
selectivity will be observed with blue-edge excitation, as a rule.
The situation is, however, different for the long-wavelength, red-edge
absorption. According to Eq. (8.45), in this case the degree of selectivity can
be greater than 1 even at In other words, if the incident light
frequency is localized at the long-wavelength edge of the absorption band,
selective excitation of nonequilibrium solvates will be ensured even when
the width of the homogeneous spectrum exceeds that of the inhomogeneous
spectrum. As seen from Eq. (8.45), however, this is true provided the
excitation frequency is within the frequency range given below:
The maximum value of the selectivity is found in the middle of this

range. On shifting toward both shorter and longer wavelengths, the excitation
selectivity decreases, and it is less than unity beyong this range. The
dependence of the selectivity on the relation between the widths of the
homogeneous and inhomogeneous spectra is illustrated for all the above cases
in Figure 8.3. Note that the degree of selectivity is also a function of the ratio
that is, it depends on how the lifetime of a molecule’s excited state
changes depending on the elementary cell configuration.
As noted above, fluctuations of the local structure become fixed in a
frozen solution and are no longer temperature-dependent (provided no phase
transitions occur with decreasing temperature in the frozen solution). There-
fore, the inhomogeneous spectrum broadening function for a solution at liquid
helium temperature is the same as at liquid nitrogen temperature and can be
calculated using Eqs. (8.21) and (8.22) for polar molecules and Eqs. (8.33)
and (8.34) for nonpolar molecules if the solution melting point is taken as the
value of T. As shown above (see Table 8.1), the width of the inhomogeneous
function has been estimated as for polar molecules and
for nonpolar molecules. These results are of the same order of
magnitude as measurements made at liquid helium temperature.(23, 26)
Thus,
for the polar 1-iodonaphthalene molecule in butyl bromide at
an inhomogeneous spectral width of was reported,(26) whereas, as
mentioned above, for the nonpolar perylene molecule in n-hexane at the same
temperature, was measured to be
Thus, the characteristics of inhomogeneous spectral broadening of
complex molecules at liquid helium and liquid nitrogen temperatures prove to
be the same. However, the homogeneous spectra of molecules are essentially
different at these temperatures. As shown in Refs. 7 and 27 for amorphous
matrices, fine-structure, rather than diffuse, absorption and fluoresencee
spectra consisting of a set of narrow nonphonon lines are observed at liquid
helium temperature. Superposition of such spectra belonging to cells of
different configurations leads to a structureless spectrum. Therefore, it is the
inhomogeneous broadening effect that plays the determining role in the
commonly observed structureless appearance of absorption and fluorescence
bands. If the homogeneous linewidth is rather small (experiments(28, 29) show
the nonphonon linewith to be to ), that is, provided the

conditions and are fulfilled, the selective excitation
function becomes symmetric about the frequency and, following Eqs. (8.45)
and (8.46), assumes the form
(8.48)
shown in Figure 8.3.
However, in reality, due to the overlap of the phonon wings at short
wavelengths, selective excitation of nonequilibrium cells at liquid helium
temperature can be realized only for long wavelengths of an inhomogeneously
broadened absorption band.
8.2.6. Absorption and Fluorescence Spectra: Dependence on Exciting Light

Frequency
Let us consider the influence of the configurational broadening on the

absorption and fluorescence spectra of a solution. To calculate the total
absorption spectrum, we will integrate the transition probability over all the
initial configurational states of the solvates and over all the initial vibrational
states of the dye molecule, taking into account the corresponding distribution
functions:
Here is the coefficient for absorption of light of frequency v by

dye molecules with the pure electronic transition frequency v el when in the
vibrational state of energy
The vibrational state-averaged Einstein coefficient
characterizes the homogeneous molecular absorption spectrum:
Also, for simplicity, let the configurational interaction be weak compared to the
intramolecular one. Then may be assumed to be independent
of the stored vibrational energy that is, the shape of the spectrum is the
same for all the solvates. Thus, the configurationally broadened absorption
spectrum can be represented as
where the distribution function is given by Eq. (8.21).

Note that such a distribution is usually observed experimentally in the
absence of strong excitation and enhanced inflow of molecules from excited
states. An increase in the exciting radiation density leads to a shortening of
the time that the molecule spends in the ground state. Thus, if the excitation
density at is such that the inequality
is fulfilled, the solvate distribution in the ground state should differ from the
equilibrium one. In this case, the solution becomes more transparent and
behaves as a spectrally inhomogeneous system.
With knowledge of the shape of the homogeneous absorption spectrum
the absorption band of the solution can be calculated from Eq.
(8.52). As shown in Ref. 30, for complex organic molecules the absorption
band can be nicely approximated by the function
The matching coefficients a and b are related to the width of the absorp-
tion band (for the level determined by the pure electronic transition
frequency ) and the distance between the absorption maximum and the
frequency by the relations
Figure 8.4 presents calculated homogeneous and inhomogeneous absorp-

tion spectra obtained from Eqs. (8.49)–(8.54) using values of the parameters
typical for phthalimide molecules. In this case, the width of the homogeneous
spectrum, is close to that of the inhomogeneous spectrum,
The configurational broadening does not, therefore, cause
any essential changes in the shape and position of the absorption band. The
deformation is most pronounced at the long-wavelength slope of the band.
The absorption in this region is mainly due to the nonequilibrium solvates. In
addition, the absorption band is transformed with temperature due to the
dependence of on T. It should be noted that with rising temperature the
broadening of the band prevails.
The steady-state fluorescence spectrum of a solution, in accordance with

Eq. (8.52), can be expressed as
where
is the Einstein coefficient for spontaneous emission integrated over vibrational

states, and is the pure electronic transition frequency distribution
function of excited cells. Using the same approximation as in Eq. (8.53), the
spectral relationship can be expressed as
where the coefficients a and b are the same as in Eq. (8.53).

As shown in Ref. 4, the relation between the configurational relaxation
time of the medium and the excited state lifetime of the molecule may
give rise to two different situations. If the condition of fast reorientation of
the solution molecules, is fulfilled, then a steady-state equilibrium
distribution of the cells with respect to configurational energy is attained
during the lifetime of the excited state. In this case, the solvate distribution is
of the following form:
The homogeneous and configurationally broadened fluorescence spectra

calculated from Eqs. (8.55)–(8.58) are also shown in Figure 8.4. In this case,
the configurational spectral broadening is seen to result in some broadening
of the fluorescence band at its short-wavelength slope. The position and shape
of the spectrum are independent of the exciting light frequency.
The situation is essentially different for a rigid solution characterized by
slow reorientation of molecules, In this case, the distribution function
depends on the exciting light frequency is the number of cells

with electronic transition frequency that are excited at excitation frequency
is the total number of excited cells.
The quantities and can be found from the balance
equations for the steady-state condition. As a result, one can easily obtain
where is the excited state lifetime for molecules with electronic transi-
tion frequency is the mean lifetime of excited molecules, and is
determined from Eq. (8.21) and from Eq. (8.53) with the excitation
frequency substituted for v.
The solution fluorescence spectrum changes with the distribution function
Figure 8.4 shows the fluorescence spectra calculated from Eqs.
(8.55)–(8.60) as a function of the exciting light frequency. Upon excitation at
the long-wavelength slope of the absorption band, the fluorescence spectrum
depends on the excitation frequency; it is shifted toward longer wavelengths
as decreases. Initially, the change in does not alter the shape of the
fluorescence band. Only for rather small incident light frequencies, that is,
upon excitation of essentially nonequilibrium solvates, is spectral broadening
observed owing to the above-mentioned decrease in the excitation selectivity
Thus, when the condition is satisfied, the configurational spectral

broadening of the solution is inhomogeneous, which is manifested as a strong
dependence of the position of the fluorescence maximum on the exciting light
frequency, the latter being localized at the long-wavelength slope of the
absorption band. The experimental verification of this conclusion was of great
importance as it could provide evidence for the validity of the theoretical
treatment.
In the next section we present results obtained from the nonhomogeneous
solution model.
8.3. Stationary Inhomogeneous Broadening
8.3.1. Universal Relationship between Fluorescence and Absorption Spectra of

Polar Solutions
Stepanov and Gribkovsky have established a general law relating the

absorption spectra of complex molecules to their fluorescence spectra.(31)
According to this law, the contours of the absorption K(v) and fluorescence
bands of such species are related by
where D(T) is a temperature-dependent constant. Equation (8.61) is analogous

to the well-known Kirchhoff law, which states the relationship between the
absorption and luminescence spectra of a blackbody, since luminescence
power is proportional to thermal emission power for complex molecules.
Sufficiently fast vibrational relaxation of molecules in both singlet states
and is the only condition for these relations to be fulfilled. As the
vibrational relaxation time in dye molecules is very short the
universal relationship should be valid for practically all solutions of complex
organic compounds. Experiments(1) have shown, however, that for polar solu-
tions the universal relationship holds at room temperature, but is violated at
low temperatures in viscous and especially in rigid frozen solutions.
It follows from the previous section that the absorption K(v) and
emission of the solution should be described as integrals over the
configurational energy continuum (Eqs. 8.52 and 8.55).
As can be seen from Eqs. (8.52) and (8.55), the relationship between the
absorption and fluorescence spectra depends on the configurational energy
continuum distribution function. Thus, if and are equilibrium
Boltzmann functions, then can easily be shown (32) to obey
Eq. (8.61). This is valid for liquid solutions at room temperature when fast
reorientation of solvent molecules occurs and the Boltzmann distribution over
configurational sublevels reaches steady state in times much shorter than the
fluorescence lifetime. If the function differs from the Boltzmann form,
which is encountered in low-temperature or high-viscosity solutions when
the reorientation is delayed or absent during the excited state lifetime, the
universal relationship of Eq. (8.61) is not obeyed.
Thus, the solution model that has been developed to account for the
inhomogeneous broadening of solution electronic spectra predicts boundaries
of validity of the universal relationship of Eq. (8.61) that are in agreement
with experimental data.
8.3.2. Luminescence Spectra at Red-Edge Excitation
As shown in Section 8.2.6, it follows from the proposed model for

polar solutions that in high-viscosity solvents (e.g., frozen ones), in which
molecular reorientation is slow or absent and energy exchange between the
configurational sublevels of the excited state is hindered or does not occur,
luminescence will occur from the configurational states into which the solvates
were excited or from the states close to them. In the most common case where
the electric dipole moment of a luminophore molecule increases on
going from the ground state to the upper singlet state, in accordance with
Eqs. (8.10), (8.14), and (8.15) and as shown on the field diagram of Figure
8.1, the steady-state fluorescence spectrum of the solution must be shifted
toward wavelengths as the viscosity of the solution increases. This
phenomenon, known as the blue S-shaped shift of the fluorescence spectrum,
has received much study for frozen solutions(1) and is most pronounced for
excitation at the absorption band maximum.
For a long time there was a firm belief that in pure dye solutions the
luminescence spectra are independent of the exciting light frequency. This
statement was formulated as a law. (33) Any deviation from this law was
attributed the presence of admixtures or aggregates in the solution.
However, it follows from the field diagram for polar solutions (Figure
8.1) that, by varying the excitation frequency, we will selectively excite
different configurational states, and in this case inhomogeneous broadening of
the solution spectra is expected.
A study of polar solution spectral properties for was undertaken.
This condition can be realized either by increasing or decreasing The
former can be achieved by freezing the solution or using polymer matrices,
whereas the latter can be achieved by quenching the luminescence by using
admixtures or a powerful light flux. AH four approaches were tested experi-
mentally.(4, 6, 34–36) The excitation frequency dependence of the fluorescence
spectrum was first revealed for frozen solutions of phthalimide derivatives.(4, 6)
Later, it was observed for rigid polymer solutions of these compounds at
room temperature as well as for other dyes.(34) As this effect consisted in the
red shift of the fluorescence band, it was called “bathochromic luminescence”
(BL). Actually, this term applies to luminescence from selectively excited
solvate states with a large store of configurational energy.
It has been shown(35, 36) that BL is also observed when the condition
is fulfilled due to the reduction of the dye molecule excited state
lifetime as a result of fluorescence quenching by impurities or in a powerful
laser field rather than due to an increase in the configurational relaxation
time of the solution. These experiments have firmly established that BL
of polar solutions is caused by inhomogeneous configurational broadening
of electronic energy levels. Later, it was found that not only singlet but
also triplet states of dyes were broadened configurationally and, under the
condition of slow reorientation of molecules in a solution, this broadening
was inhomogeneous.(37)
The application of tunable dye lasers for excitation opens up new
possibilities of studying configurationally nonequilibrium solvates in a
solution. Figure 8.5 gives BL measurement data for a frozen ethanol solution
of the well-known dye rhodamine 6G under fluorescence excitation by a
continuous-wave (cw) tunable dye laser. The solid curve (I) represents the
absorption spectrum. At the bottom of the figure, a set of luminescence curves
corresponding to excitation at different wavelengths (indicated by the arrows)
in the far anti-Stokes spectral region is given. Absorption in this region is
already very small due to the small population of the upper configurational
states. Nevertheless, the excitation of luminescence proved to be possible due
to the high laser radiation intensity employed. Excitation at frequency
near the absorption maximum yielded the fluorescence spectrum labeled 1.
The position and the shape of this spectrum are practically independent of
the frequency for varying within the main part of the absorption
band, where the broadening is largely due to the vibrational sublevels of the
equilibrium unexcited configurational state. The situation changes, however,
when is shifted to the anti-Stokes region. Because of the abrupt break in
the long-wavelength slope of the absorption spectrum of complex molecules in
solution, there exists a possibility of selective excitation of some solvate

groups whose electronic transition frequencies differ from that of the equi-
librium configurational state. It should be noted that with decreasing an
appreciable red shift (up to several tens of nanometers) of the luminescence
spectrum is observed. In liquid solutions at room temperature, this effect dis-
appeared and the luminescence band did not depend on
Figure 8.5 also shows the BL excitation spectrum obtained by recording
rhodamine 6G fluorescence at a fixed wavelength, In essence, this
spectrum characterizes the absorption of nonequilibrium solvates of a given
configuration (determined by the wavelength at which the fluorescence is
recorded). It is seen that the excitation spectrum is similar in shape to the
ordinary absorption spectrum, but it is substantially red-shifted. This is
consistent with the suggestion that the two spectra belong to identical dye
molecules which differ only in configuration.
Thus, as follows from the model representations and experimental results
reported in this section and Section 8.2.6, it is possible to selectively excite and
study solvates of essentially nonequilibrium configuration whose fluorescence
spectrum is shifted to lower frequency by many hundreds of wave numbers
relative to the ordinary fluorescence spectrum. The information on such non-
equilibrium solution microstructures is of particular interest because cells of
abnormally large local electric field intensity are concerned here. In such cells
the dye molecules may differ not only in their spectral frequencies, but, in
principle, also in their nonradiative transition probabilities.(38)
8.3.3. Directed Nonradiative Energy Transfer in Organic Solutions
It has been known for many years that the influence of inductive-
resonance energy transfer between similar molecules on the luminescence
properties of rigid solutions is restricted by the fact that the degree of
polarization, the quantum yield, and the decay time decrease with increasing
concentration. (39) The last two effects are believed to be caused by energy
migration to low-luminescence centers(39); that is, they would not be observed
if luminescence centers of only one type were present in the solution. The
majority of previous theories which described the influence of energy transfer
on the luminescence characteristics of solutions (see Refs. 39–41) did not
predict the existence of any concentrational effects besides those mentioned
above.
The presence of a continuous set of configurational solvate states presup-
poses the possibility of inductive–resonance energy transfer between different
states provided that the concentration of dye molecules in the solution is high
enough. If such a transfer does occur, it will apparently be directed from the
solvates with a high frequency of the pure electronic transition to those with
lower values of this frequency, that is, from the “blue” solvates to the “red”
ones (the reverse transfer must be hindered due to its low probability).
All this is illustrated by the diagram in Figure 8.6 of electronic–configura-
tional states in solution. For simplicity, the diagram depicts a discrete, rather
than a continuous, set of electronic–configurational states.
When the solution concentration is increased, the sublevel may be deac-
tivated by both spontaneous emission and nonradiative energy transfer
(dashed lines in Figure 8.6) to solvates with lower frequencies of
the pure electronic transition, since the probability of energy transfer in this
direction is higher than in the reverse direction because of different overlap
integrals of the absorption and emission spectra of “blue” and “red” solvates.
For rigid solutions, gradual passage to excitation on the low-frequency slope
of the absorption spectrum, that is, decreasing the excitation frequency,
results in selective excitation to the first singlet state of the solvates that, in
their ground state, populate configurational sublevels higher than the equi-
librium sublevel (Figure 8.6). When excited, these solvates will, accordingly,
populate the configurational sublevels lying below the Franck–Condon sub-
level I* (up to the equilibrium sublevel II*). At the same time, the efficiency
of nonradiative energy transfer decreases, since energy transfer to more
“red” solvates practically does not occur due to their insignificant proportion
in the solution, and the reverse energy transfer to the sublevel I* is of low
probability.
Directed nonradiative energy transfer (DNRET) in rigid polar solutions
and polymer matrices is responsible for a number of peculiar concentrational
effects. These include the long-wavelength shift of luminescence spectra with
increasing luminophore concentration(13) and the dependence of emission
anisotropy on the luminescence and absorption spectra.(42) For the emission
spectra, luminescence anisotropy is higher on the blue slope and drops toward
longer wavelengths. In the case of red-edge excitation, the emission anisotropy
values increase to close to the limiting values.
It was also found that in solutions of complex molecules with inhomo-
geneously broadened spectra, at high concentrations some unusual time
characteristics of the luminescence were observed: the luminescence decay
time increased toward longer wavelengths, and the luminescence decay on the
slopes differed appreciably from single exponential.(38, 42) Finally, it has been
shown (43) that for red-edge excitation the concentrational effects disappear in
systems with inhomogeneously broadened spectra, as follows from the
diagram in Figure 8.6.
DNRET was directly observed for the first time using nanosecond
spectrofluorimetry. This technique makes it possible to visualize the dynamics
of the process and measure the required kinetic parameters. (44) In Ref. 45,
DNRET was observed between different configurational states of solvated
molecules in a rigid solution. Poly(vinyl alcohol) was used as the solvent.
At a low dye concentration, steady-state inhomogeneous broadening was
observed: for 3-aminophthalimide molecules the luminescence spectrum
shifted as the exciting light frequency was varied. In other words, in this case
ordinary bathochromic luminescence was observed, consistent with selective
excitation of various configurational states of solvated molecules. The batho-
chromic luminescence spectrum was time-independent. At high concentrations
(Figure 8.7) the situation was, however, different. The instantaneous emission
spectrum shifted gradually to the red. In this case, each instantaneous
spectrum corresponds to a specific solvate state whereas the entire sequence
of spectra represents the energy transfer dynamics for a set of configurational
states. The higher the dye molecule concentration, the faster is the energy
transfer. As would be expected, the energy transfer direction is always the
same, from the “blue” to the “red” solvates. The time shift of the luminescence
spectrum is responsible for the nonexponential luminescence decay in the
emission spectrum, being most distinct on its slopes. In addition, the lumines-
cence decay time depends on the recording wavelength (Figure 8.7, curve 5).
Figure 8.8 shows the position of the luminescence spectrum maximum versus
time for solutions with different concentrations of 3-aminophthalimide. In line
with theory, the rate at which the spectrum shifts is seen to depend on time.
It is maximum just upon excitation and then decreases. With decreasing
concentration, the time shift amplitude decreases.
The unique feature of directed energy transfer in the systems involved is
that it occurs between chemically identical molecules with different shells
rather than between chemically different molecules with differently located
energy levels. This means that in this case the directed transfer results in
spontaneous concentration of excitation energy on molecules with a specific
surrounding structure, that is, in some structurally specified local regions of the
rigid solution. Of particular interest is the fact that energy is transferred from
structures with a low degree of molecular orientation to those characterized
by a high degree of orientation. Indeed, the dependence of the electronic
transition frequency of a molecule on the local field intensity is defined by
Eq. (8.10). This suggests that the higher the solvate reactive field intensity,
the lower is the transition frequency. In other words, the “blue” centers
correspond to solvates with low R, that is, with a more chaotic orientation of
solvent molecules in the solvate shell, whereas the “red” centers represent
solvates with a high-intensity reactive field and, therefore, with a higher
degree of orientation of their molecules.
Thus, the migration of electronic excitation from “blue” to “red” centers
can be regarded as directed energy transfer from structures of low order to
highly ordered structures in the solution. Such a process can naturally be
expected to play a certain part in the mechanism of directed energy transfer
in biological systems, in particular, in the transfer of electronic excitation
energy from the antenna chlorophyll molecules to the reactive center in the
photosynthetic system of plants. In Refs. 19 and 20 energy exchange between
molecules of the photosynthetic pigments chlorophyll a and pheophytin a was
studied experimentally under model conditions with pigments introduced into
the polar matrix. Data for chlorophyll a are presented in Figure 8.9. Both the
instantaneous spectrum and the spectrum provide evidence for the existence
of DNRET in this case.
We conclude by noting that the theory of inductive–resonance energy
transfer in solutions with inhomogeneous spectral broadening is given in Refs.
17 and 42.
8.4. Dynamic Inhomogeneous Broadening in Liquid Solutions
8.4.1. Analysis of Configurational Relaxation in Liquid Solutions
In liquid solutions, unlike the rigid systems discussed in Sections 8.2.6

and 8.3.2, the optically excited nonequilibrium distribution function of solvates
is easily converted into the equilibrium one due to the high rate of inter-
molecular energy exchange. This process must involve the variation of the
light frequency with time, that is, a shift of the solution fluorescence spectrum
during emission. The verification of these conclusions was of great interest to
provide support for the theoretical model of the dynamic nature of configura-
tional inhomogeneous spectral broadening in liquid polar solutions. Special
equipment was designed to selectively excite individual inhomogeneous
broadening states and to record the luminescence with the required time
resolution.(11)
Let us consider the basic luminescence characteristics of a liquid solution.
Substitute the values of and as functions of the excitation parameters
and time into the expression for luminescence power (Eq. 8.55):
Equation (8.62) is rather general as it takes into account not only the
change with time and the dependence of the excited state population on the
experimental conditions, but also the dependence of the spontaneous emission
spectrum on these conditions.
Analysis of Eq. (8.62) reveals all the experimental features of lumines-
cence of polar solutions including the kinetics of the instantaneous emission
spectra [at a given /, we have the instantaneous spectrum for a given instant
of time, and the decay law if the frequency is fixed
and t is variable.
8.4.1.1. Instantaneous Spectra

As follows from Eq. (8.62), the configurational relaxation of luminescence
spectra can be described if we know the type of distribution function that
describes the distribution of solvates over the configurational broadening
states. It is not clear, however, how this function can be calculated in the
general case from the model considerations. Assuming weak dependence of
on Eq. (8.62) can be rewritten as
where is the half-width of the distribution function. It is seen from

Eq. (8.63) that the process can be described approximately by taking into
account the frequency relaxation corresponding to the maximum of the
function
In Ref. 22 the analytical expression for configurational relaxation of an
excited solvate is substantiated. Let us consider its derivation. Excitation of a
solution at a radiation frequency involves selective excitation of solvates
with the local field intensity determined from Eq. (8.10). The excited
solvates are reoriented, with tending to due to the total unbalanced
force (see Section 8.2.1) and the friction force which may be assumed
to be proportional to the rate of change of the reactive field during relaxation:
If
substituting Eqs. (8.4) and (8.64) into Eq. (8.65) and integrating over t, we
obtain
Expressing the electric field intensities in terms of frequencies of the exciting

light and the corresponding solvate transitions using Eqs. (8.10) and (8.14),
and taking into account the relationship between the frequencies and
according to Eq. (8.15), one can obtain the molecular electronic transition
frequency as a function of time and exciting light frequency:
Assuming for simplicity that the absorption and fluorescence spectra are
mirror images and taking into account that for the cells with field R0 to be
selectively excited it is necessary to irradiate the solution with radiation of
frequency rather than determined from Eq. (8.67), we obtain
the explicit form of the dependence of the solvate fluorescence maximum on
time and the exciting light frequency
Let us rewrite this expression in a somewhat different form:
where is the deviation of the solvate fluorescence maximum at time t

from its equilibrium position In Eq. (8.68),
that is, is the maximum absorption frequency of solvates with local

field
As seen from Eq. (8.69), at the initial instant of time linearly
depends on the exciting light frequency; that is, inhomogeneous spectral
broadening is present. Over time, becomes less dependent on that is,
the inhomogeneous broadening decreases, and as it vanishes.
Thus, for liquid solutions the degree of inhomogeneous broadening
decreases with time; in other words, it is dynamic in nature. As the
fluorescence maximum tends to its equilibrium value, determined by the
frequency
The relaxation of the fluorescence spectrum depends essentially on the
exciting radiation frequency, that is, on the type of selectively excited solvates.
The following three cases are possible:
1. Excitation in the Stokes spectral region results in a red

shift of the fluorescence spectrum with time. We
call (10,11) such a process the configurational down-relaxation of the
luminescence spectrum. This type of relaxation can easily be

explained in terms of molecular reorientation in solvates of similar
configurations in the ground electronic state, and it was experi-
mentally observed long ago.(46, 50, 51)
2. For excitation in the anti-Stokes spectral region the situa-
tion is the reverse; that is, at the initial instant of time the spectra are
red-shifted as compared to the steady-state spectra In this
case, the return of the spectrum to its normal position during con-
figurational relaxation will lead to a blue shift with time. From the
physical point of view, this means that the configurational interaction
energy excess which solvates possess during relaxation is partially
converted into emitted quantum energy, which just leads to an
increase in the radiation frequency with time. In this connection, the
process has been termed the “up-relaxation” of the luminescence
spectra.
3. Finally, the third case corresponds to pumping of the solution by the
frequency In this case, the luminescence spectrum immediately
after excitation must be close to the steady-state one and will
not vary with time. From the physical point of view, this case
corresponds to the situation where those solvates are excited whose
configuration is nonequilibrium in the ground state, but corresponds
to the equilibrium configuration in the excited state (i.e., solvates
with a local field It should be noted that the frequency
simultaneously characterizes both the spectral properties of the dye
molecules and the effect of inhomogeneous broadening; that is, it is
a complex characteristic of a solution. It separates the excitation
frequency ranges responsible for “down-” and “up-relaxation” of the
fluorescence spectra and is called the equilibrium configurational
excitation frequency. (22)
As seen from Eq. (8.69), the relaxation rate of the fluorescence spectrum
is determined by the value of If the friction coefficient is a constant,
then the decay obeys a simple exponential law. In a more general case, of
course, the coefficient must depend on the rate of change of the reactive
field, dR/dt, and increase with it. This, in turn, must inevitably lead to
more complicated decay laws which can be described by assuming to be
a function of R or by replacing the exponent in Eq. (8.69) by a sum of
exponents with corresponding coefficients, as was done in
Ref. 46.
Note that the nature of all the implications of Eq. (8.69) can be
qualitatively explained using the field diagram of a solvate.
The relationship is calculated from Eq. (8.69) for a typical
phthalimide molecule in n-propanol at different temperatures in Ref. 11.
We think that the advantage of the proposed model is its independence

of any particular diagram of polar solution energy levels, as none of them was
used to derive Eq. (8.69).
8.4.1.2. Fluorescence Decay

For configurational orientation at the laws of spontaneous
emission decay as well as the instantaneous spectra are determined by the
evolution of the distribution function of the solvates, and, hence, they
depend on the whole set of parameters which determine the inhomogeneous
broadening function,
To analyze the decay laws, it is necessary to specify the shape of the
fluorescence contour and its variations with pumping frequency and time. The
contour will be described, as before, in terms of the hyperbolic function [see
Eq. (8.57)], but we will assume that the frequency shifts with
time according to Eq. (8.69). Let the decrease in the total population of all
conformational states obey a simple exponential law,
and originate from its initial value of given by the excitation

power. As in the previous section, we will study the decay law in the regions
of down- and up-relaxation and at their boundaries.
Let us consider the case of Stokes excitation and assume, for simplicity,
that It can easily be shown that, under such conditions, in the
short-wavelength range of luminescence frequencies the fluorescence decay is
defined by the expression
that is, the decay is faster than without relaxation. In the long-wavelength
spectral range,
which is indicative that the configurational relaxation is associated with an

increase in the fluorescence decay time. Finally, in the region of the maximum
of the equilibrium fluorescence spectrum
that is, the fluorescence decay time does not vary due to the configurational
relaxation. The above types of behavior are shown in Figure 8.10a (curve 1).
It is quite clear, from the physical point of view, that the drift of the
fluorescence spectrum away from the recording frequencies is equivalent to
a decrease in whereas the shift of the spectrum toward the recording
frequencies must lead to an increase in and even rise of luminescence intensity

provided the relationship between the parameters is favorable. The latter effect
was predicted in Ref. 48. However, it has not yet been clearly observed in the
nanosecond region.
The situation is quite different for excitation in the far anti-Stokes region
of the absorption spectrum; beyond the equilibrium configuration excitation
frequency, the fluorescence decay time will be described not by Eq. (8.73), but
by Eq. (8.72), whereas for the low-frequency portion of the spectrum,
is specified by Eq. (8.72) rather than by Eq. (8.73). However, in the region of
the maximum of the equilibrium spectrum the decay law is not distorted by
relaxation, as before. These results are illustrated by curve 3 in Figure 8.10a.
Light pumping at the equilibrium configuration excitation frequency
yields a purely exponential decay law (Eq. 8.74) for all regions of the
fluorescence spectrum, since in this case the fluorescence spectrum does not
undergo a shift with time (Figure 8.10a, curve 2).
Summarizing the results of the analysis of the spectral behavior, it is
necessary to describe three different types of dependences of the fluorescence
decay for variation of the excitation frequency over a wide range. Such a
diversity of spectra is due to the existence in the solution of different
types of solvates, each having its own dynamic luminescence spectral
behavior, and the possibility of their selective excitation by monochromatic
laser light. If this fact is neglected, the picture obtained is poor and describes
the solvation shell reorientation kinetics for only one type of centers. This
was the situation that existed before the concepts of the configurational
broadening of polar solution spectra were introduced. The fluorescence decay
behavior in a viscous homogeneous solution was considered for the first
time in Ref. 48 in terms of the Onsage model under the assumption that
the maximum shift of the fluorescence spectrum due to the reorientation
of the molecules of the solvation shell followed an exponential law. The
authors calculated the curves from the emission spectrum, assuming
instantaneous excitation, and showed their essential dependence on the
recording frequency.
Somewhat later, identical results were obtained using both a discrete and
a continuous model of the solution.(49)
All the theoretical dependences considered in this section have been
verified and supported by the methods of time-resolved spectroscopy. The
results are given in the next section.
8.4.2. Experimental Study of the Luminescence Kinetics of Liquid Solutions
The study of the time shift of the spontaneous emission spectrum with
varying energy of the exciting radiation is of indisputable interest, since it
enables the conclusions about the process of intermolecular relaxation that
have been made on the basis of the model presented here to be verified.
The first comprehensive measurements in both the Stokes and anti-
Stokes regions were reported in Refs. 10 and 11. The studies were performed
with a nanosecond spectrofluorimeter (time resolution about 1 ns) using a
tunable pulsed dye laser as the excitation source.
Figure 8.11 gives the instantaneous fluorescence spectra (panel a) and

the position of their maxima at different times (panel b) as functions of the
excitation frequency for 3-ANMP. It is seen that a decrease in the excitation
frequency leads to a reduction in the fluorescence spectrum range
Thus, for whereas at
Thus, the frequency corresponds
approximately to the equilibrium configurational excitation frequency
Further decrease of reverses the behavior of the relaxational spectral shift;
that is, the shift of the spectrum with time is toward shorter wavelengths.
Characteristically, in all cases the position of the maximum in the emission
spectra relaxes to that in the steady-state fluorescence spectrum (Figure 8.11b,
dashed line) recorded at room temperature, whereas at short times after
excitation the position of the maximum in the instantaneous emission spectra
strongly depends on the excitation frequency. The above result is a direct
manifestation of inhomogeneous configurational broadening of electronic
spectra of complex molecules in viscous solutions.
It is worth noting that in the course of spectral relaxation the shape of
the instantaneous fluorescence spectra of fluorescent solutions is little affected
by the transformation of the inhomogeneous broadening function with time.
This is illustrated in Figure 8.11a, which shows instantaneous fluorescence
spectra with excitation in the Stokes region. At the position of the
fluorescence spectra can be assumed to correspond to that at low tem-
perature, that is, under conditions of slow molecular reorientation.
The experimental spectral/kinetic behavior in Figure 8.11a and b agrees
with the theoretical relationships shown in Figure 8.11c. The results obtained
were observed for cooled, polar solutions in which the orientational relaxation
is delayed. Exactly the same spectral behavior was observed(52) for low-
viscosity liquid solutions at room temperature, in which the orientational
relaxation rate is much higher. A 6-ps-resolution spectrometer was used in the
experiments.
Thus, it has been shown that the difference in behavior between
instantaneous fluorescence spectra of different groups of inhomogeneous
solution centers is indicative of the existence of dynamic inhomogeneous
configurational broadening in polar liquid dye solutions. Previously, only
long-wavelength shifts of emission spectra with time were observed under
configurational relaxation with Stokes excitation by a continuous pulse-
discharge spectrum (50,51) and a fixed nitrogen laser frequency. (46) The short-
wavelength shift of fluorescence spectra (up-relaxation) with time is a radically
new effect which was observed for the first time in Ref. 10. Us treatment
necessarily involves inhomogeneous dynamic broadening of the solution
spectra. The experiments have revealed the existence of reorientation of excited
solvate molecules due to the disturbance of the electrical equilibrium in the
electronic transition.
Interesting results have been obtained for the luminescence lifetime

spectra presented in Figure 8.10b for a viscous 3-ANMP solution in n-propanol
at different excitation frequencies It is seen that on excitation of dye
molecules in the Stokes region, the luminescence lifetime monotonically
increases with decreasing frequency from 2.5 ns to
20.5ns for 3-ANMP solutions at
With decreasing excitation frequency, the lifetime spectrum is modified. The
range of sharp increase of on the frequency scale is decreased and shifts
toward shorter wavelengths. Beginning at some excitation frequencies (at
which up-relaxation already appears), qualitatively different spectra
are observed. These are characterized by a monotonic increase in with
increasing Thus, for excitation of 3-ANMP at 21,190 cm–1 the value of
is 4 ns at v rec = 16,670 cm–1 and increases to 16.5 ns at v rec = 21,740 cm–1
The above features of the behavior of lifetime spectra of viscous solutions
with variation in the excitation frequency are in good agreement with the
configurational relaxation model considered in the previous section. The
field diagram of the energy levels (Figure 8.1) can also contribute to their
qualitative explanation.
To conclude, it should be noted that simultaneous configurational
relaxation of excited levels and their spontaneous deactivation results in non-
exponential fluorescence decay. The rate of change of the intensity decreases
monotonically with time at the high-frequency edge and increases at low
frequencies for excitation in the Stokes region with The reverse is
observed at Only for is the decay rate constant over the
time.
8.4.3. The Solution Spectrochronogram
As mentioned above, it is not clear how the relaxation of the distribution

function can be calculated in the general case from a model.
However, to correctly describe the luminescence kinetics, it is necessary to
take into account that the spectrochronogram when excited by
a pulse, represents convolution of the solvate distribution function over
frequencies of the 0–0 electronic transition, and of the homogeneous
luminescence spectrum, in agreement with Eq. (8.62). Equation
(8.62), which assumes an identical lifetime for all sublevels, can be rewritten
in a more convenient form for practical applications as
where is a constant.
As shown in Section 8.2.3, the equilibrium inhomogeneous function of a

polar solution is Gaussian in shape, which permits us to write it for ground
and excited states as
Here the subscripts g and e indicate the ground and the excited singlet state,
respectively; and are the root-mean-square deviation of this
distribution and the position of its maximum during relaxation, respectively.
Since the width of the homogeneous absorption spectrum is large due to
vibrational broadening, it is impossible to excite selectively only one type of
solvates. Therefore, immediately after optical excitation the instantaneous
luminescence spectrum is inhomogeneously broadened. Since the number of
solvates with the 0–0 transition frequency at the zeroth instant of time in the
excited state is equal to the product of the number of solvates in the ground
state with the same frequency times the absorption coefficient of this type of
solvate, the inhomogeneous broadening function at time t = 0 is equal to
where is the exciting light frequency, is the homogeneous

absorption spectrum, and is the equilibrium distribution function in
the ground state, which can be found from Eq. (8.52).
Equations (8.75)–(8.76) and (8.52) make it possible to find the functions
and exactly, as well as the solution correlation function
where is the position of the
center of gravity of the distribution function at different times.
Figure 8.12 shows the instantaneous luminescence spectra of 3-ANMF in
glycerin calculated at time t = 0 (curve 1) and experimentally recorded at
times t = 3 ns (curve 2) and t = 16 ns (curve 3), as well as the inhomogeneous
broadening functions (curve 4) and (curves 5 and 6) for
these same times; has been calculated from Eq. (8.77), and
was found by deconvolution of instantaneous luminescence spectra recorded
on a laser spectrometer.
The steady-state luminescence and absorption spectra of 3-ANMF in
nonpolar n-hexane have been taken as the homogeneous spectra. The calcula-
tions show that, with the assumptions of a Gaussian shape, is about
at t = 0, then increases to (t = 3 ns), and decreases again
to (t =16 ns)
As follows from Eq. (8.75), the fluorescence kinetics at a fixed frequency
are determined by the variation of the inhomogeneous broadening function
with time, which, in turn, depends on the functions and Calcula-
tions using Eq. (8.75) have shown that experimental and calculated decay
curves are well matched (Figure 8.13) if is specified as
where are constants.

Since in the initial stage of relaxation the luminescence kinetics are
mainly determined by the variation of the inhomogeneous function, for the
sake of simplicity of calculation can be given by one exponential form
with a certain relaxation time.
Thus, for liquid polar solutions the fluorescence spectrochronogram
is well described by a continuous model if we associate changes in the
half-width of instantaneous spectra with the evolution of the inhomogeneous
broadening function of the solution and express its relaxation as the sum of
two exponents.
8.4.4. The Effect of Light-Induced Molecular Rotation in Solution
The configurational relaxation which takes place upon excitation in

systems with dynamic inhomogeneous spectral broadening not only manifests
itself in a shift of the instantaneous luminescence spectra and a change in the

decay law over the spectrum, but it also affects the rate of Brownian
molecular rotation of the luminophore in solution. This effect depends on the
initial store of configurational energy of solvates in an excited electronic state,
which, in turn, is determined by the excitation frequency, and can, therefore,
be considered as light-induced rotation of molecules in the liquid (LIR). LIR
leads to the dependence of the depolarization behavior of the solution
luminescence on the excitation and recording frequency. (21)
If the solvates are excited at a frequency and the fluorescence emission
is recorded at then in the course of relaxation the solvates giving rise to
the recorded emission have decreased their potential energy by (Figure
8.1). This energy is mainly converted into rotational energy of solvation shell
molecules. The released energy can be estimated from the magnitude of the
shift of the instantaneous solution fluorescence spectra. For 3-ANMP in
glycerin excited at its absorption maximum, this shift is about
Taking into consideration that the solvate includes one 3-ANMP molecule
and five or six glycerin molecules, each solvate molecule accounts for
of energy released during the configurational relaxation. This value
is comparable to the thermal energy of molecules in solution at room
temperature. The fluctuational force acting on the solute molecules increases
as the average rotational energy of the neighboring solvent molecules is
increased. This must accelerate the luminescence depolarization of the
solution upon excitation by linearly polarized light.
Characterizing the depolarization due to the release of the excess energy
by the value of and assuming its linear relationship with we have
Using Eqs. (8.10) and (8.11) and neglecting the vibrational spectral
broadening (in this case, we can write this expression as
where is a constant determined by the properties of the

solution.
Taking into account the fluorescence depolarization due to optically
induced molecular rotations, the emission anisotropy is
where is the emission anisotropy in the absence of intermolecular relaxation.

Let us analyze the following three characteristic cases. In the first case,
the solution is excited near the absorption band maximum and
Then, it follows from Eqs. (8.79) and (8.80) that with decreasing
increases and the emission anisotropy r decreases.
Thus, in the case of Stokes excitation, the fluorescence depolarization due
to configurational relaxation is most pronounced at the red edge.
When the solution is excited in the anti-Stokes region (configurational

up-relaxation), Then, it follows from Eqs. (8.79)
and (8.80) that increases and decreases with increasing Hence, the
accelerated fluorescence depolarization is more pronounced at the blue edge.
Finally, when the solution is excited at the frequency of zero confi-
gurational excitation and In this case, the
excitation does not induce molecular rotation.
The above model considerations have been confirmed by experiment. (21)
Different solutions (mainly, of phthalimide derivatives) in glycerin and normal
alcohols as well as phospholipid bilayer membranes were investigated using a
nanosecond laser fluorimeter. The experiments revealed that in systems in
which the configurational relaxation occurs on a time scale comparable to the
excited state lifetime, the fluorescence depolarization rate depends on the
recording frequency. In addition, the emission anisotropy kinetics (EAK) are
characterized by two components: a fast one in which the measured rate of
decrease of the emission anisotropy depends on the excitation and
recording frequencies, and a slow component which is independent of
the experimental conditions.
The experiments also revealed that with excitation at the absorption
band maximum, the decrease in the emission anisotropy with time is more
rapid at the long- than at the short-wavelength edge. The red-edge decay is
multiexponential (Figure 8.14a). In this case, the instantaneous fluorescence
spectra undergo a long-wavelength shift (Figure 8.14b). It is also seen that the
dependence of the instantaneous anisotropy values on the recording
wavelength becomes stronger during the course of configurational relaxation.
The relaxation energy decreases with decreasing excitation frequency. The
dependence of the EAK on thus weakens with decreasing excitation
frequency and, therefore, the instantaneous values of anisotropy vary less
rapidly with changes in With excitation of the solution at the frequency
of zero configurational excitation the EAK are no longer dependent on
the recording frequency.
Further decrease of the excitation frequency gives rise to a
short-wavelength shift of the instantaneous spectra (up-relaxation)(10) (Figure
8.15). As a result, the blue-edge EAK are multiexponential (Figure 8.15a), and
the instantaneous anisotropy spectra (Figure 8.15b) are the reverse of those
for The values of the molecular rotation times measured from double
exponential fit of anisotropy decay for different excitation and recording
wavelengths are presented in Table 8.2.
The experimental data confirm that the random-force impulses rotating
the luminophore molecules during relaxation following excitation depend on
the released configurational energy of the solvate. In the course of relaxation,
the excess potential energy due to dipole–dipole interactions of solvate
molecules is expended in their rotational degrees of freedom. It should be
noted that this additional kinetic energy imparted to the molecules turns out
to be significant and is sufficient to rotate them by a particular angle. The
value of this angle depends on the intermolecular solvate energy after optical
excitation. Such an effect, as mentioned above, can be regarded as light-
induced molecular rotation.
The emission anisotropy additivity rule has been used to estimate the
angle of rotation of the luminophore molecule.(53) According to this rule, if
there are several types of luminescent centers in the system, the emission
anisotropy of the whole system is equal to the sum of the values of the
emission anisotropy of the individual components. If the emission only from
the Franck–Condon and equilibrium sublevels is taken into account (Figure
8.la), this rule can be written as
where the characterize the emission anisotropy kinetics for the Franck–
Condon (i = 1) and equilibrium (i = 2) states, and is the parameter charac-
terizing the contribution to the total fluorescence of the ith state, which is
determined by the expression
where the characterize the decay kinetics for the Franck–Condon and
equilibrium states calculated from the differential balance equations for the
populations of the states, the are the spectra of the states, and
is the integrated luminescence intensity of both types of states.
As in the case of spherical top type molecules, the emission anisotropy

kinetics of the individual states can be represented by
where are the limiting values of the emission anisotropy of the

states, is the Fitting parameter for the decrease in emission anisotropy
during configurational relaxation due to the light-induced rotation of solvate
molecule, and are the times of Brownian rotation of molecules in
the different states.
Equation (8.81) was used to estimate the value due to the effect of
light-induced molecular rotation. The calculation was performed for 3-ANMP
in glycerin with experimentally determined parameters. The spectrum of
3-ANMP in hexane, which does not display inhomogeneous broadening,
served as the spectrum of the Franck–Condon state The equilibrium
state spectrum was taken as the instantaneous spectrum of 3-ANMP in
glycerin after configurational relaxation.
The experimental results were fitted for both Stokes (up-relaxation) and
anti-Stokes (down-relaxation) excitation.
Figure 8.16 gives the EAK calculated for excitation of the solution at the
absorption band maximum. Comparison with the experimental data (Figure
8.12) shows that satisfactory agreement takes place at
Using the Levshin-Perrin formula (54) for the limiting value of the
emission anisotropy,
where is the angle between the absorption and emission oscillators, one can
determine the angle of light-induced rotation of the luminophore molecule
during configurational relaxation. For 3-ANMP in glycerin, this value was
at the excitation frequency
8.5. Selective Kinetic Spectroscopy of Fluorescent Molecules in

Phospholipid Membranes
8.5.1. Energy Levels of an Electric Dipole Probe in a Membrane
The phospholipid bilayer (Figure 8.17) which makes up a membrane is

such that the hydrophilic parts of the phospholipid, the charged heads, project
outward, whereas the hydrophobic parts of these molecules, the hydrocarbon
chains, lie inside the bilayer. By virtue of such organization, the membrane
has a significant polarity gradient—the polarity decreases as one moves from
the periphery to the center of the bilayer. On embedding a probe, the outer
part of the membrane, with the probe incorporated into it, behaves as a polar
solvent and the inner part as a nonpolar solvent.
Many probe molecules, such as aminonaphthalenes and phenylnaphthyl-
amines, have an electric dipole moment and are located in the membrane close
to its polar region.(55) Since the incorporation of a probe into the membrane
is a statistical process, the probe is distributed throughout the membrane
thickness in a specific manner. This leads to a distribution of the energy of
intermolecular, configurational probe–environment interactions. Furthermore,
the individual energies in this distribution fluctuate due to the thermal motion
of the probe and, hence, its different orientations relative to the local field, as
well as due to the segmentary motion of the membrane. As in the case of
polar solutions, the statistical distribution of the probe–membrane interaction
energy, according to Eq. (8.10), must cause inhomogeneous broadening of the
luminophore’s electronic spectra.
The probe luminescence lifetime, which usually ranges from nanoseconds
to tens of nanoseconds, can be used as the natural time scale. At room
temperature the rotation time of the probe in the membrane is comparable to
its luminescence lifetime whereas the radial motion of the probe, that is, its
translational motion, is much slower. The inhomogeneous spectral broadening
for the probe due to its localization at different depths in the membrane must,
therefore, be static, as in the case of rigid polar solutions. The broadening due
to fluctuations in the probe–environment interaction energy at a given depth
will be dynamic, as in the case of viscous solutions.
Let us construct, by analogy with polar solutions, a diagram of electronic-
configurational states of the system consisting of the probe molecule and its
immediate surroundings in the membrane (we shall call this system the
solvate). Let us plot on the abscissa the local electric field intensity R and on
the ordinate the solvate potential energy, which includes the electronic energy
of the probe and the probe–environment interaction energy as well as the
interaction energy of the solvate membrane segments.
Such a diagram for two different probe localization depths is shown in
Figure 8.18, where the left-hand pair of curves corresponds to the most
probable localization. On each curve the energy minimum corresponds to the
Inhomogenaous Broadening of Electronic Spectra of Dye Molecules 415
most stable configuration of the probe and the solvate membrane segments,
for which all the types of interaction are in relative equilibrium. Thermal
motion can also give rise to other solvate configurations with lower or higher
values of R. In this case, however, the relative equilibrium of intermolecular
forces is disturbed and the potential energy of the solvate increases.
As the solvate configuration and, therefore, the field do not change
during the transition of the probe molecule to another electronic state, the
electronic transitions in Figure 8.18 are represented by vertical lines (inter-
molecular treatment of the Franck–Condon principle). In the excited state
the electric dipole moment generally increases. Thus, in the upper state
the potential energy minima are shifted toward higher R as compared to in
the ground state. It can be shown that, as in the case of solutions, the pure
electronic transition frequency of the probe in the membrane is unambiguously
related to the distance between the combining pairs of configurational sub-

levels in the ground and excited states.
Upon transition to another electronic state, the equilibrium in the solvate
is disturbed due to the change in the probe dipole moment. Therefore, as soon
as the probe molecule finds itself in another electronic state, the process of
configurational relaxation and establishment of the equilibrium Boltzmann
distribution over configurational sublevels take place. As can be seen from
Figure 8.18, upon excitation at the absorption spectrum maximum or close to
it, this process involves a decrease in the emitted energy.
The spectral properties of the entire system consisting of the membrane
and the probe molecules should be analyzed using the diagram in Figure 8.18
and taking into account the distribution of the membrane solvate over the
different states associated with the different depths of localization of the
luminophore as well as the distribution (at each depth) over the states caused
by local field fluctuations.
When analyzing the diagram shown in Figure 8.18, one should bear in
mind that at room temperature no transitions are observed between differently
localized centers because of the low velocity of the translational motion of
the probe during the excited state lifetime. Therefore, at room temperature the
process of configurational relaxation that occurs within several nanoseconds
mainly involves orientational motion of the probe and, to some extent,
segmentary reconstruction.
8.5.2. Inhomogeneous Broadening in Steady-State Fluorescence Spectra of

Probes
As mentioned above, one of the characteristic features of inhomogeneous

broadening of electronic spectra of complex organic compounds in condensed
media is the long-wavelength shift of the luminescence spectrum upon
excitation by narrow-band radiation at the red edge of the absorption
spectrum. Such an effect has been observed for dipolar fluorescent probe
molecules in a number of cases(59) in both polar solutions and membranes.
For 1-phenylnaphthylamine (1-PNA), bathochromic fluorescence is illustrated
in Figure 8.19, which shows experimental data for the position of the emission
band in solutions (Figure 8.19a) and in membrane phospholipid bilayers
(MPB) (Figure 8.19b) as functions of the excitation wavelength. A significant
shift of the luminescence spectrum toward longer wavelengths upon red-edge
excitation is observed for both rigid and viscous glycerin solutions and in
lecithin liposomes. It is noteworthy that the spectral shift does not involve any
substantial deformation of the emission bands. There are no isosbestic points
or any other effects which could be ascribed to chemical or quasi-chemical
bonds between the luminophore and the environment.
The bathochromic fluorescence shift of the probe in MPB can easily

be interpreted in terms of the diagram given in Figure 8.18. With red-edge
excitation, mainly solvates with low values of the 0–0 transition frequencies
are promoted to the upper singlet state. As configurational relaxation occurs
on the nanosecond time scale, that is, on a time scale comparable to the
excited state lifetime of the probe, the emission for each probe localization
depth is from the same state as or a similar state to that in which the solvate
found itself after excitation. This causes a bathochromic fluorescence shift with
decreasing energy of the exciting radiation.
The influence of the membrane viscosity on the observed effect is

demonstrated by measurements of the position of the fluorescence spectrum
for 1-PNA in liposomes as a function of the temperature and the excitation
wavelength (Figure 8.20). For edge (390-nm) excitation, the position of the
spectrum slightly depends on temperature. When the membrane is heated,
the positions of the spectra for ordinary and edge excitation come closer
together because of the accelerated configurational relaxation in the system.
The temperature dependence of the position of the fluorescence maximum
(Figure 8.20) is complicated. The monotonic fluorescence maximum versus
temperature curve has a plateau in the temperature range 20 to 40 °C. This
effect can apparently be attributed to the complex nature of the inhomo-
geneous broadening of the electronic spectra of the probes in the membrane.
It is not improbable that the position of the fluorescence spectrum maximum
in the range shifts only due to the rotational dynamics of
the phospholipid segments and the probe and that, beginning at the
fluorescence spectrum maximum is affected by the translational motion of the
probe. The plateau of the fluorescence maximum versus temperature curve
from 20 to indicates that the configurational relaxation of the probe
and membrane segments is already sufficiently fast and no longer affects the
position of the spectrum of the solvate located at a certain depth. At the same
time, within this temperature range the role of the translational motion of the
probe in mixing states which differ in depth within the membrane is not
significant.
8.5.3. Kinetics of Probe Fluorescence
An understanding of the elementary mechanisms of the complex processes

by which the numerous functions of biological membranes are realized
involves detailed information not only on the molecular structure of the latter,
but also on their structural dynamics. It is therefore necessary to elucidate the
kinds and the characteristic times of motions of the membrane molecules and
segments.
Time-resolved spectroscopy makes it possible to study the dynamics of
configurational relaxation, which are manifested by shifts in the instantaneous
fluorescence spectra upon optical excitation.
Studies of the fluorescence decay curves, anisotropy, and nanosecond
time-resolved fluorescence spectra(56) of 2-PNA in lecithin liposomes revealed
the existence of nanosecond dynamics. However, the mechanism of probe
rotation was not specified but was assumed to be equilibrium Brownian
diffusion in nature. Later, phase fluorometry was used (57) to observe the
spectral kinetics of 1-PNA.
In Ref. 58, the fluorescence spectra of 1-PNA in liposomes measured on
a nanosecond fluorometer were reported to show shifts toward longer
wavelengths on the nanosecond time scale (Figure 8.21). The magnitude of
the shift decreases as the excitation wavelength increases. However, even with
410-nm excitation, for which a significant bathochromic shift of the steady-
state fluorescence spectra is observed, the time dependence of the instan-
taneous emission spectra was observed (2–8 ns after the excitation pulse there
occurs a shift of the instantaneous spectra by 10 nm). In our opinion, these
data can easily be interpreted in terms of the model considered here as
follows.
Upon excitation at 337 nm, all types of fluorescence centers at the different
localization depths pass to the excited state. After excitation, configurational
relaxation takes place, during the course of which each type of fluorescence
centers tends toward its equilibrium configuration.
As is clear from the field diagram (Figure 8.18), the nanosecond spectral
dynamics are associated with the configurational nonequilibrium of the
solvates. Their existence even at 410-nm excitation indicates that the distribu-
tion function of the solvates in 0–0 transition frequency, which is associated
with the static inhomogeneous broadening, decreases toward longer
wavelengths less rapidly than does the distribution function associated with
the dynamic inhomogeneous broadening.
Directly associated with the long-wavelength shift of the probe emission
spectrum with time is the dependence of the fluorescence lifetime on the wave-
length (see Figures 8.22 and 8.23). The parallel processes of configurational
relaxation and deactivation of the excited state lead to essentially non-
exponential decay kinetics for the luminescence at the red and the blue edge
of the spectrum.
8.5.4. Rotational Dynamics of the Probe in the Membrane
Let us now direct our attention to the diagram of energy levels of fluo-
rescent centers in a membrane (Figure 8.18). In the case of excitation such that
(the subscript i indicates that the frequency of zero configurational
excitation is different for centers with different depths of localization), energy
relaxation begins upon excitation. During relaxation, the excess configura-
tional energy is expended in other degrees of freedom of the fluorescent
center. As in the case of LIR in solution, this must lead to an increase in the
fluctuation force acting on the probe and acceleration of the process of
fluorescence depolarization. It is clear that the effect is the stronger, the
greater the intermolecular interaction energy released in the course of
relaxation. This conclusion is confirmed by experiment.
The studies reported in Ref. 58 have shown that the emission anisotropy
kinetics of 1-PNA in MPB essentially depend on the excitation and recording
wavelengths. For excitation within the long-wavelength absorption band the
kinetics are nonexponential, and the depolarization rate decreases with time
(Figure 8.23). The red-edge fluorescence depolarization is always faster than
the depolarization at the blue edge.
With excitation in the region of the maximum of the long-wavelength
absorption band (337 nm), the moment the excitation pulse causes, the
emission anisotropy values are significantly lower than the limiting value. This
points to the existence of a subnanosecond component of the depolarization
that is invisible under the experimental conditions. With excitation at the red
edge of the absorption band, (390 nm, Figure 8.23), the values of the emission
anisotropy increase practically up to the limiting value; that is, the subnano-
second component of the depolarization disappears.
The data indicate that for 1-PNA molecules in phospholipid membranes
the process of configurational relaxation of the luminescence spectrum,
which follows the process of excitation, accelerates the probe fluorescence
depolarization. This depolarization is most pronounced when the recording
frequency is at the red edge of the fluorescence spectrum, where the greatest
contribution to the luminescence is made by solvates that have undergone
relaxation.
The increase in the fluorescence anisotropy on going to longer excitation
wavelengths (Figure 8.23) may be attributed to the lower configurational
energy of the solvates absorbing the lower frequency radiation.
The dependence of the luminescence depolarization rate on the recording
wavelength influences the character of the steady-state polarization spectrum;
that is, as the wavelength increases, the depolarization monotonically
decreases. The wavelength dependence of the fluorescence decay time also
essentially contributes to this effect.
As indicated above, light-induced rotation was also observed in polar
solutions of some phthalimide derivatives. Therefore, we may maintain that

this phenomenon is universal, and it should be taken into account when
considering the spectroscopic and photochemical properties of different systems
which include polar molecules that exhibit inhomogeneous configurational
broadening of their electronic spectra. The specific character of excitation-
induced probe rotation in a membrane is associated with the nature of the
membrane structure, in particular, with the great length of phospholipid
molecules. Thus, the light-induced rotation of the probe in a membrane will
be more pronounced than in a solution with smaller, more mobile solvent
molecules.
The dependence of the probe rotation rate in a membrane on the
configurational energy of the luminescent center complicates the problem of
determining the microviscosity of membranes from the Levshin–Perrin
formula, since this formula was derived for diffusion in a system at thermo-
dynamic equilibrium without consideration of the influence of nonequilibrium
solvation in a polar solution molecular rotation. It is clear that the rotational
diffusion time from which the microviscosity is calculated should be determined
from experiments unaffected by the light-induced rotation of the probe.
Furthermore, one should bear in mind that in the case of edge excitation we
determine the microviscosity of that portion of the polar membrane where
most red fluorescent centers are located.
The value of for 1-PNA in a phospholipid membrane calculated from
Using the procedure described in Section 8.4.4, the angle of rotation of

1-PNA in a membrane undergoing configurational relaxation was found to be
about 22°.
8.6. Conclusions
We will summarize here the results presented in this chapter. The data
that have been obtained to date enable us to draw the following conclusions:
1. Polar solutions of complex molecules are systems with inhomo-
geneous configurational broadening. The differences in the spectra
of the fluorescent centers in a polar solution are caused by the
variability in structure of the solvation shells surrounding the fluo-
rescent molecule owing to the thermal motion of the molecules in the
solution.
2. In rigid solutions, inhomogeneous broadening is static in nature and
shows up in steady-state spectra. Static inhomogeneous broadening
is observed in liquid solutions, too, when the condition of slow
molecular reorientation is satisfied. This condition can be
fulfilled by reduction of the luminescence lifetime under strong

quenching by impurities or under powerful light.
3. In liquid solutions, inhomogeneous configurational spectral broad-
ening is dynamic in nature. This is manifested, in particular, by the
dependence of the position of the instantaneous fluorescence spectra
on the excitation frequency and the time at which the spectra are
recorded. The dependence on the excitation frequency decreases with
time, and at the spontaneous spectra no longer depend on
the excitation frequency.
For polar solutions there exists a special parameter, the equi-
librium configurational excitation frequency of the solution, This
parameter corresponds to the excitation frequency at which the
quasi-equilibrium distribution of excited solvates over configurational
states subsequent to excitation is attained.
4. The difference between the solvates in a solution is manifested in
wavelength dependences of the decay time as well as of the depolar-
ization kinetics of fluorescence. The rotation rate of luminophore
molecules is found to depend on the configurational energy of the
solvate. This explains the change of the rate of fluorescence depolar-
ization with the wavelength of excitation.
5. Inhomogeneous broadening is pronounced in electronic spectra of
dipolar fluorescent probe molecules in biological membranes, where
it is mainly associated with the heterogeneous character of the
localization of the luminophore in the bilayer. This inhomogeneous
broadening is responsible for the bathochromic shift of the fluo-
rescence band of the probe molecule and its temperature dependence,
the kinetics of instantaneous fluorescence spectra, and light-induced
rotation of probe molecules.
The discovery of inhomogeneous configurational spectral broadening of

solutions and the study of its fundamental properties not only proved to be
of great utility in spectroscopy of organic liquid and frozen (300 to 77°K)
solutions with broad-band spectra, but it has also aided in the understanding
of nonphonon transitions of the same molecules at liquid helium temperatures.
In the latter case, as indicated in Sections 8.2.3 and 8.2.5, inhomogeneous
broadening is of the same physical nature as at liquid nitrogen temperature.
It can be stated with confidence that the notions about inhomogeneous
configurational broadening are of practical importance not only for spectro-
scopy, but also for other sciences. These notions are already being used to
study the structure and structural dynamics of biopolymers,(59) which is
extremely important in elucidating the functional properties of biological
molecules, since the latter are determined, to a great extent, by these factors.
The concept of inhomogeneous broadening requires that the interpretation of
existing data be revised from new standpoints, and it promises fundamentally

new results.
In chemistry, consideration of nonequilibrium solvation is of particular
importance in kinetics, since solvation conditions have a strong influence on
the rates and mechanisms of chemical reactions. By modifying the solvation
conditions by selective optical excitation, it is possible to manipulate the rates
of chemical reactions.(60)
Acknowledgment
The results presented in Section 8.5 have been obtained in collaboration

with Prof. A. P. Demchenko and Dr. N. V. Shcherbatskaya from the Institute
of Biochemistry, Kiev, and Dr. D. M. Gakamsky from the Institute of
Physics, Minsk.
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Index
Actomyosin, 140 Collisional quenching, 53; see also Quenching

ADAS: see Anisotropy decays, decay-associated Confidence intervals, 207
spectra Monte Carlo, 214
Adrenocorticotropin, 33
Alcohol dehydrogenase, 110 Data analysis
Analysis: see Least-squares analysis anisotropy, 22
Anisotropy, 1 global, 241
fiber optics, 362 least-squares, 113, 177
fundamental, 7, 138 quenching, 112, 193
oriented systems, 307 Diffusion
quenching-resolved, 105 light-induced, 406
wavelength-dependent, 318 rotational, 8
Anisotropy decays, 1, 407 two-dimensional, 96
associated, 28, 283, 411 Distance distributions, 157
data analysis, 22 acceptor decay, 278
decay-associated spectra (ADAS), 284 frequency domain, 162
definition, 2 global analysis, 277
electron spin resonance, 331 myosin, 168
ellipsoids, 9 quenching-resolved, 160
frequency domain, 26 theory, 158
global analysis, 280 troponin I, 166
hindered rotation, 15 Distributions, 261; see also Distance
instrumentation, 25 distributions
membranes, 287 distance, 271
multiexcitation wavelength, 286
muscle fibers, 325 Ellipsoids, 9
order parameter, 17 Energy transfer, 127, 261
oriented systems, 320 actomyosin, 140
proteins, 32 applications, 140
quenching-resolved, 105 distance distribution, 157
Associated anisotropy decay, 29; see also efficiency, 130
Anisotropy decay enzyme kinetics, 152
Azurin, 102 Förster distance, 129
inhomogeneous broadening, 390
Broadening, 367; see also Inhomogeneous labeling, 133
broadening lanthanides, 148
myosin, 134
Calmodulin, 48 orientation factor, 131, 135
Italic numbers indicate a detailed description of the topic.

429
430 Index
Energy transfer (Cont.) Global analysis (Cont.)

quenching-resolved, 160 quenching, 112
ribosomes, 147 Glucagon, 108
stopped-flow, 153
time-resolved, 155 Horse liver alcohol dehydrogenase, 110
troponin, 145, 166
tyr-to-trp, 150 Immunoglobulin, 44
Error analysis, 207, 288; see also Data analysis Inhomogeneous broadening, 367
Excited state reaction, 272 intensity decay, 399
membranes, 413
F-Actin, 43 steady-state emission, 416
Fiber optics stationary, 387
light sources, 359 time-resolved emission spectra, 396
mode, 347 vibrational broadening, 376
transmission, 349 Instrumentation, anisotropy decays, 25
Fiber-optics sensors, 345
design, 349 Labeling, oriented systems, 332
mode, 347 Lanthanides, 148
polarization, 362 Lifetime-resolved anisotropy, 105
time-resolved, 361 Least-squares analysis, 177
transmission, 349 assumptions, 181
Fluorescence sensors, 345; see also Fiber-optic chi-square test, 233
sensors confidence intervals, 207
Förster distance, 129 cumulative frequency plots, 223
Frequency domain Durbin–Watson test, 231
anisotropy decays, 27 frequency domain, 199
distance distributions, 162 Gauss–Newton, 187, 203
global analysis, 253 global analysis, 258
intensity decays, 26 implementation, 235
troponin I, 166 Kolmogorov–Smirnov test, 233
FRET, 127; see also Energy transfer Neldes–Mead, 193, 205
outliers, 228
Gauss–Newton algorithm, 187 quenching, 113, 199
Global analysis, 241; see also Least-squares residuals, 216
analysis; Data analysis Simplex, 193, 205
anisotropy decay, 280 theory, 180
associated anisotropy decay, 283 trends, 226
decay-associated spectra, 284 Lifetime distributions, 114, 261
distance distributions, 277 Light sources, fiber optics, 359
eigenvalue/eigenvector, 267 Lumazine, 37
error analysis, 288, 294
excited state reaction, 272 Melittin, 33, 107
flow chart, 259 Membranes, 92, 413, 422
frequency domain, 253 surface potential, 99
identifiability, 288 Micelles, 92
implementation, 248 Microscopy, oriented systems, 313
intensity decay, 252 Monellin, 108
least-squares, 258 Muscle fibers, anisotropy decay, 325
multidye, 285 Myelin basic protein, 108
multiexcitation wavelength, 286 Myosin, 41, 134
overview, 244
physical models, 261 Naphthol, excited state reaction, 274
Index 431
Nelder–Mead Simplex algorithm, 193 Quenching (Cont.)

Nitroxide quenching, 98 electrostatic effects, 71
Nucleic acids, 100 energy transfer, 108, 160
Nuclease, 108 frequency domain, 88, 199
heterogeneous systems, 58
Order parameter, 17, 331 least-squares analysis, 113
Orientation factor, 131, 135 lifetime distributions, 114
Oriented systems, 307 ligand binding, 75
electron spin resonance, 331 mechanisms, 78
instrumentation, 313, 319 membrane-bound proteins, 93
labeling, 332 membranes, 92, 96
order parameter, 331 membranes and micelles, 92
time-resolved, 320 micelles, 92
nitroxide, 98
Perrin equation, 137 nucleic acids, 100
Phosphorescence, 117 phosphorescence, 117
Polarization, 5, 307; see also Anisotropy; proteins, 68
Anisotropy decays resolution of lifetimes, 103
Proteins resolution of spectra, 102
actomyosin, 140 quenchers, 67
adrenocorticotropin, 33 quenching-resolved, 105
alcohol dehydrogenase, 110 radiation boundary, 61
azurin, 102 Smoluchowski, 60
calmodulin, 48 static, 64
F-actin, 43 Stern-Volmer, 55
glucagon, 108 surface potential, 99
immunoglobulin, 44 transient effects, 60, 87
lumazine, 37
melittin, 33 Red-edge excitation, 388, 417
membrane-bound, 93 Resonance energy transfer, 127; see also Energy
monellin, 108 transfer
myelin basic protein, 108 Ribosomes, 147
myosin, 41, 134, 140, 168 Rotational diffusion, 8
nuclease, 108 correlation time, 31
quenching, 68 ellipsoids, 10
red-edge excitation, 388, 417 S. nuclease, 32
resolution of spectra, 102 Segmental flexibility, 18
ribosomes, 147 Selective excitation, 378, 383
S. nuclease, 32, 88 red-edge excitation, 388
single tryptophan, 69 vibrational levels, 378
troponin, 146, 146 Sensors, 345; see also Fiber-optic sensors
troponin I, 166 Smoluchowski quenching, 60; see also
Quenching
Quenchers, 67, 93 Solvent effects, inhomogeneous broadening, 367
interaction with proteins, 85 Solvent relaxation
partitioning, 92 inhomogeneous broadening, 395
Quenching, 53 intensity decay, 399
anisotropy, 105 Static quenching, 64
conformational changes, 76 Stern-Volmer, 53; see also Quenching
data analysis, 199 Stopped-flow fluorescence, energy transfer, 153
distance distribution, 160
efficiency, 59 Time-resolved anisotropy, 1
432 Index
Time-resolved energy transfer, 155 Transient effects, 87

Time-resolved emission spectra, inhomogeneous Troponin, 145, 146
broadening, 396, 420 Troponin I, 166
Total internal reflectance, fiber optics, 346
Transfer RNA, 39 Yt-base, 40

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KLUWER ACADEMIC PUBLISHERS

©2002 Kluwer Academic Publishers

All rights reserved

Created in the United States of America

Visit Kluwer Online at: http://www.kluweronline.com

Katalin Ajtai • Department of Biochemistry and Molecular Biology, Mayo

Nicolai A. Nemkovich • Institute of Physics of the B.S.S.R. Academy of

1. Fluorescence Anisotropy: Theory and Applications

1.6.4. Anisotropy Decay of Calmodulin Complexes with TNS . . . . 48

2. Fluorescence Quenching: Theory and Applications

2.7. Phosphorescence Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

3. Resonance Energy Transfer

4. Least-Squares Analysis of Fluorescence Data

4.5.1.Example of the Gauss–Newton Procedure . . . . . . . . . . . . . . . 202

5. The Global Analysis of Fluorescence Intensity and Anisotropy Decay

5.4. In-Depth Flow Chart of a General-Purpose Global Analysis

6. Fluorescence Polarization from Oriented Systems

7. Fluorescence-Based Fiber-Optic Sensors

8. Inhomogeneous Broadening of Electronic Spectra of Dye Molecules in

8.5. Selective Kinetic Spectroscopy of Fluorescent Molecules in

Fluorescence anisotropy decay belongs to the general class of relaxation

Robert F. Steiner • Department of Chemistry and Biochemistry, University of

1.2.1. Meaning of Anisotropy

The radiation emitted by a fluorophore may be polarized to varying

dinate axes are designated The sum of these three components

The exciting beam may correspond to either unpolarized, or natural, light or

so that the total intensity is given by, from Eq. (1.1),

Finally, if the exciting beam is linearly polarized along the Ox axis, we

It is usual to perform fluorescence anisotropy measurements with an

In terms of the usual laboratory arrangement, may be identified

and, for unpolarized exciting radiation,

For horizontally polarized exciting light, we have

If more than one fluorescent species is present, the observed anisotropy is

where are the emission anisotropy of species i and its fractional

However, since the use of anisotropy leads to simpler expressions, it has

1.2.2. Influence of Excitation Pulse Shape

Observations of the time decay of fluorescence intensity or anisotropy

where is the intensity at zero time, corresponding to the time of the

1.2.3. The Time Decay of Anisotropy

The measurement of fluorescence anisotropy depends upon the selective

where are the components of the molecular absorption and

where are the direction cosines of the linear transition moments of

When a sample containing a population of fluorophores whose orienta-

attached to the macromolecule, with a fixed orientation of its transition

1.2.4. The Rotational Diffusion of Ellipsoids of Revolution

The rate of rotational diffusion may be characterized by a rotary diffusion

Here, W is a probability density function describing the distribution of

where k is Boltzmann’s constant, T is the absolute temperature, is the

In order to describe the hydrodynamic properties of proteins, it is often

It is also useful to define a set of three rotational correlation times,

For a particle with spherical symmetry,

Introducing the value of

1.2.5. The Anisotropy Decay of Ellipsoidal Particles

1.2.5.1. Theoretical Treatment of Anisotropy Decay

Here, D (the mean rotational diffusion coefficient)

or 312) where are the cosines of the angles formed by the

In the limiting case of a particle with spherical symmetry, for which

where For a symmetrical ellipsoid of revolution, Eq. (1.29)

For an ellipsoid of revolution for which the absorption or emission

For the case of an ellipsoid of revolution for which the transition

where is the rotational diffusion coefficient about the axis of symmetry,

1.2.5.2. Simulation of Anisotropy Decay