Tools & Techniques

AFM Principles and

Application in Biosciences
Mr. R. Rajasekaran

Abstract
In recent year, Atomic Force
Microscope (AFM) has provided a
range of now opportunities for viewing,
Manipulating and analyzing
biomolecules in the environments. And
will hopefully allow application to be
developed for AFM in Medicine and
biotechnology.
Keywords:Atomic Force Microscope
(AFM), Biomolecules, Interaction tip,
Piconewton (10-12N).
Introduction
The invention of ATOMIC FORCE
MICROSCOPY (AFM) in the mid-1980s6,
followed by continuous progress in
instrumentation, (AFM). A relatively new
form of microscopy in which a sharp tip is
scanned over the surface of a sample, while
sensing the interaction force between the tip
and the sample. Because AFM does not rely
on an incident beam, as in electron or light
microscopy, the specimen can be directly
observed at high Resolution in aqueous
solution.
The newly developed atomic force
microscope is a valuable tool for studying
physical and biological structures provides a
unique window to the microworld of cells,
subcellular structures, and biomolecules. The
AFM can image the three-dimensional
structure of biological specimens in a
physiological environment.
This enables real-time biochemical and
physiological processes to be monitored at a
resolution similar to that obtained for the
electron microscope. Atomic force
microscopy since the initial reports of
considerable progress has been made, but
applications in the field of biology are
exploring biological structures under
conditions in which living organisms exist.
AFM is a kind of scanning probe microscope
where imaging of the sample is realized by
interaction of the probe with the sample
surface and no imaging beam (light or
electron) is involved in the process. The tip of
the probe is mounted on the end of a flexible
cantilever (1).
Sample preparation and recording conditions,
has revolutionized the way in which
microscopists explore biological structures.
This surface imaging technique involves
scanning a sharp tip over the surface of a
sample, while sensing the interaction force
between the tip and the sample of biological
specimens at sub-nanometre resolution under
physiological conditions.
In fact, an atomic force microscope is more
than just a microscope as it can also measure
minute forces within or between biological
molecules a method known as Force
spectroscopy (Box 1) in a way that was not
previously possible the last decade of using
AFM and related Scanning Probe Microscopy
techniques in biology showed that their
popularity and power continue growing.
Numerous reviews both comprehensive and
specialized cited in the reference list and
articles devoted to biological applications of
Atomic Force Microscopy prove this fact (2).
Box - 1
Force spectroscopy
A form of AFM in which the force acting on
the tip is measured with piconewton
(10-12 N) sensitivity as the tip is pushed
towards the sample. Then retracted from it.
The Principles of Atomic
Force Microscopy
Atomic force microscopy (AFM) is a member
of a family of new microscopic techniques
that are referred to as scanning-probe
microscopes (SPMs). The concept on which
all SPMs are based is the generation of images
of surfaces by measuring the physical
interaction between a sharp tip and the sample
rather than by using an incident beam (light or
electrons) as in classical microscopy. The
main parts of an atomic force microscope are
the sample stage, the CANTILEVER ( Box 2)
and the optical detection system, which
comprises a laser diode and a hotodetector.
The sample is moved relative to the cantilever
in three dimensions using PIEZOELECTRIC
CERAMICS (Materials that expand or
contract when subjected to a potential
difference.) The force interacting between the
tip and the sample is monitored with
piconewton (10-12N) sensitivity, by attaching
the tip to a soft cantilever, which acts as a
spring, and measuring the bending (or
deflection) of the cantilever (Fig-1). The
cantilever deflection is usually detected by a
laser beam focused on the free end of the
cantilever and is reflected into a photodiode
(2). AFM cantilevers and tips are usually
made of silicon or silicon nitride using
MICROFABRICATION techniques ( Box .3)
Box - 2
Cantilver
AFM tips are mounted on cantilever beams or
triangles, which are typically made of silicon
or silicon nitride, that behave like springs.
Using Hooke's law, the magnitude of the
tipsample force is proportional to the
deflection of the cantilever.

38 | Advanced Biotech | June 2008


Fig: 1. Atomic force microscopy.
Box - 3
Microfabrication
A range of techniques that are derived from
the techniques used in Microelectronics to
make integrated circuits and which are used to
make AFM tips and cantilevers.
The different operating
modes of Atomic Force
Microscopy
Atomic force microscopes can be operated in
various modes in the constant-force imaging
mode, images are created by bringing the tip
and sample into contact and scanning the tip
across the surface while the sample height is
adjusted using a feedback loop to keep the
bending or DEFLECTION (The vertical
bending of the AFM cantilever resulting from
the tipsample interaction force) of the atomic
force microscope cantilever constant. This
yields a topographic image that gives
calibrated height information about the
sample.
In many cases, small cantilever deflections
occur because the feedback loop is not
perfect, and the resulting error signal can be
used to generate a deflection image, In
addition to being used as a microscope, an
atomic force microscope can measure
biomolecular forces with piconewton
(10-12N) sensitivity (3). In this mode known
as force spectroscopy the cantilever
deflection which is useful for revealing
surface details of corrugated samples.
39 | Advanced Biotech | June
In tapping mode atomic force microscopy
(TMAFM), an oscillating tip is scanned over
the surface and the amplitude and phase of the
cantilever are monitored near its resonance
frequency. As the tip touches the sample
surface only at the very end of its downward
movement, lateral forces during imaging are
greatly reduced, which is advantageous for
imaging 'soft' biological samples.
This is recorded as the tip is pushed towards
the sample and retracted from it. Using
appropriate corrections, a force versus
separation distance curve is obtained. Such a
curve can be exploited to gain insights into a
variety of surface properties and molecular
interactions and to manipulate single
molecules. Importantly, force distance curves
can be recorded at multiple locations of the
(x, y) plane to yield spatially resolved maps of
properties and interactions.
Advanced methodologies
for sample preparation
Little sample preparation is required for
bioimaging with the AFM. In most cases it is
as simple as spotting a few microliters of
solution on mica or glass. Of course
contaminations that cover surface features
have to be avoided or removed. First the
substrate-adsorbate should be rinsed with a
large excess of buffer. The following
procedures are dialysis, centrifugation and
homogenization. In order to get good contrast
and to reduce mechanical damage of the soft
biological materials, the samples can be
stabilized by adding covalent cross-linking
agents or certain cations that are able to link
2008
Tools & Techniques
the constituents of the sample to each other or
to the substrate. Cooling can also stiffen the
sample. Nevertheless, all these methods have
significant influence on the properties of
biomolecules. The sharper tip now is
available commercially and really does help a
lot.
To observe biological structures in their
native state by atomic force microscopy
(AFM), samples must be firmly attached to a
solid support to resist the lateral forces
exerted by the scanning tip. Several
immobilization strategies have been
established for microbial specimens. For
isolated membrane proteins, the most
frequently used procedure is based on
physical adsorption on a flat support, such as
mica, glass or silicon oxide, in the presence of
the appropriate electrolytes. For whole cells,
however, immobilization by means of simple
adsorption procedures is generally
inappropriate because the contact area
between the cells and the support is very
small, often leading to the cell being detached
by the scanning tip. To solve this problem, air-
drying and chemical fixation can be used to
promote cell attachment, but these treatments
can cause significant denaturation of the
s p e c i m e n . A l t e r n a t i v e l y, g e n t l e r
immobilization procedures have been
developed in which the cells are mechanically
trapped either in an agar gel or in a porous
membrane (4), In the latter method, a
concentrated cell suspension is gently sucked
through an isopore polycarbonate membrane
with a pore size that is slightly smaller than
that of the cell. This simple approach can be
used to image single, spherical, bacterial,
yeast and fungal cells under aqueous
conditions, while minimizing denaturation of
the surface molecules.
Biological Applications of
AFM
One of the advantages of AFM is that it can
image the non-conducting surfaces. So it was
immediately extended to the biological
systems, such as analyzing the crystals of
amino acids and organic monolayers.
Applications of AFM in the biosciences
include DNA and RNA analysis; Protein-
nucleic acid complexes; Chromosomes;
 Tools & Techniques
Cellular membranes; Proteins and peptides;
molecular crystals; Polymers and
biomaterials; Ligand-receptor binding. Bio-
samples have been investigated on lysine-
coated glass and mica substrate, and in buffer
solution. By using phase imaging technique
one can distinguish the different components
of the cell membranes.
There has been recent success imaging
individual proteins and other small molecules
with the AFM such as collogen. Employing
selective affinity binding procedures has
successfully imaged smaller molecules that
do not have a high affinity for common AFM
substrates. Thiol incorporation at both the 5'
and 3' ends of short PCR products has been
shown to confer a high affinity for ultra flat
gold substrates. A similar approach was used
to immobilize antibodies (IgG1) on treated
mica. In this case, the low affinity that IgG
molecules have for mica was overcome by
cloning a metal-chelating peptide into the
carboxyl terminus sequence of the IgG's
heavy chain. The recombinant sequence was
transformed into cells that expressed the
complementary light chain. The purified IgG
containing the metal-chelating peptide was
shown to bind in a regiospecific manner to
nickel-treated mica. Covalent binding of
biological structures to derivatized glass
substrates has also enabled high resolution
imaging of some samples that are not stable
on untreated glass substrates. New
approaches in AFM have provided a solid
foundation from which research is expanding
into more complex analyses. Higher
resolution imaging of a variety of small
molecules is improving at a rapid pace.
The recent innovation, such as Digital
Instruments BioScope system, which
combines the high resolution of AFM with the
ease of use and familiarity of inverted optical
microscopes, has further added to the
attractiveness of AFM for biological imaging.
Bright-field, flourescence and other optical
techniques can be used to identify structures
of interest while the AFM simultaneously
generates nanometer-resolved images of the
sample surface. (3, 5, & 9)
Application in Cell Biology
Cell biologists have applied the AFM's unique
capabilities to study the dynamic behavior of
living and fixed cells such as red and white
blood cells, bacteria, platelets, cardiac
myocytes, living renal epithelial cells, and
glial cells. For example, plasma membrane in
migrating epithelial cells has been imaged in
real time. The dynamic membrane
invagination process was observed in the
presence of calcium and when calcium levels
were reduced the process was prevented.
30nm lipidic pore formation could also be
resolved during calcium reduction. AFM
imaging of cells usually achieves a resolution
of only 20-50 nm, not sufficient for resolving
membrane proteins but still suitable for
imaging other surface features, such as
rearrangements of plasma membrane or
movement of submembrane filament
bundles. The requirement for the imaging
buffer is not restrictive, as long as the buffer
does not severely affect the integrity of the
cells. EDTA should be avoided because cells
will detach from the substrate in the absence
of divalent cations. Cultured cells normally
adhere well to the substrate and are not
displaced by modest probe forces (Fig-2).
Fig: 2. Protein Surface layers.
The AFM has been used to image living cells
and the underlying cytoskeleton, chromatin
and plasmids, ion channels, and a variety of
membranes. Dynamic processes such as
crystal growth and the polymerization of
fibrinogen and physicochemical properties
such as elasticity and viscosity in living cells
have been studied. Nanomanipulations,
including dissection of DNA, plasma
membranes, and cells, and transfer of
synthetic structures have been achieved
(6 & 9).
40 | Advanced Biotech | June 2008
Application in Microbiology
The AFM has been used to viewing and
analyzing the ultra structure of microbial cell
surface studies and it is used to investigated
the property of structure include to analyzing
structure of native membrane proteins at sub-
nanometre resolution, Function-related
conformational changes in single proteins,
Surface ultra structure of living cells, Cell-
surface dynamics, and Morphology of
biofilms. The physical properties and
biomolecular interactions such as Stiffness of
cell walls, Local surface charge and
h y d r o p h o b i c i t y, E l a s t i c i t y a n d
conformational properties of single
molecules, Mechanical stability of
supramolecular assemblies, Unfolding
pathways of membrane proteins, Molecular
forces determining cell adhesion and cell
aggregation also analyzed (2, 7 & Fig-3).
A
B
Fig-3
a. Three-dimensional AFM image of
Saccharomyces cerevisiae
Yeast cell immobilized in a porous membrane.
b. High-resolution deflection image of the surface
of Phanerochaete chrysosporium Fungal
Spores.
Application in Nucleic acid
research
One area of significant progress is the
imaging of nucleic acids. The ability to
generate nanometer-resolved images of
unmodified nucleic acids has broad biological
applications. Chromosome mapping,
transcription, translation and small molecule-
DNA interactions such as intercalating

mutagens, provide exciting topics for high-
resolution studies. The first highly
reproducible AFM images of DNA were
obtained only in 1991. Four major advances
that have enabled clear resolution of nucleic
acids are: Control of the local imaging
environment including sample modification;
Tapping Mode scanning techniques;
Improved AFM probes (such as standard
silicon nitride probes modified by electron
beam deposition and Oxide Sharpened
NanoProbes) and Compatible substrates
(such as salinized mica and carbon coated
mica) (8).
Application in Biomolecular
Interaction Studies
Along with direct imaging of biological
objects Atomic Force Microscopy plays a
significant role among numerous biophysical
methods for investigation of specific and non-
specific molecular interactions such as DNA
replication, protein synthesis, protein-
protein, enzyme-substrate, antigen-antibody,
receptor-ligand and drug-target association's
interaction, and many others - are largely
governed by intermolecular forces. AFM has
the ability to measure forces in the
nanonewton range. This makes it possible to
quantify the molecular interaction in
biological systems such as a variety of
important ligand-receptor interactions.
Another application of AFM force
41 | Advanced Biotech | June
measurements is to image or quantify
electrical surface charge. The dynamics of
many biological systems depends on the
electrical properties of the sample surface. In
addition to measuring binding forces and
electrostatic forces, the AFM can also probe
the micromechanical properties of biological
samples. Specifically, the AFM can observe
the elasticity and, in fact, the viscosities of
samples ranging from live cells and
membranes to bone and cartilage (10). AFM
gives scientists a key tool to investigate the
real world.
Conclusion
Now applications of AFM probing is far
beyond of these approved ones. It is found to
be useful in pharmacology, biotechnology,
microbiology, structural biology, molecular
biology, genetics and other biology related
fields.
Suggested Reading
1. T. Guha, R. Bhar, V. Ganesan, A. Sen, R. L.
Brahmachray, 2000. Application of AtomicForce
microscopy in seed surface studies, Current
Science journal, Vol.78, No.11, pp.1294-1295.
2. Yves F. Dufrêne, 2004. Using Nanotechniques to
explore microbial surfaces, Nature Review
Microbiology, Vol.2, pp.451-460.
3. Morris, V. J., Kirby, A. R. & Gunning, A. P. Atomic
Force Microscopy for Biologists (Imperial College
Press, London, 1999).
2008
Tools & Techniques
4. Kasas, S. & Ikai, A, 1995. A method for anchoring
round shaped cells for atomic force microscope
imaging, Biophysics Journal, Vol. 68, pp.1678-
1680.
5. Shao, Z., Mou, J., Czajkowsky, D. M., Yang, J. &
Yuan, J.Y, 1996. Biological atomic force
microscopy: what is achieved and what is needed,
Adv. Phys, Vol. 45, pp.186.
6. Jena, B. P. & Hörber, J. K. H, 2002. Methods in Cell
Biology, Vol. 68, 3350 (Academic Press, San
Diego).
7. Pum, D. & Sleytr, U. B, 1995. Monomolecular
reassembly of a crystalline bacterial cell surface
layer (S-layer) on untreated and modified silicon
surfaces, Supramol. Sci, Vol. 2, pp.193197.
8. Clausen-Schaumann, H., Seitz, M., Krautbauer, R.
& Gaub, H. E, 2000. Force spectroscopy with
single bio-molecules, Curr. Opin. Chem. Biol, Vol.
4, pp.524530.
9. W ww.chembio.uoguelph.ca/educmat/chm729/
afm/applicat.htm Hong-Qiang Li: Biological
Applications of AFM (1997)
1 0 . W w w. s p m t i p s . c o m / b i b l i o g r a p h y /
biology/introduction SPM Applications in Biology
About the Author
R. Rajasekaran.
East Street, Sirupuliyur,
Thirupalanam (PO), Thiruvaiyaru (TK).
Thanjavur (DT), Pin- 613204. TN
Phone : 04362-261187.
Mobile: 9943511685
E-mail:rrs_rajesh2004@yahoo.com