Flourescence quenching quantum mechanics and measurement of

quenching flourescence

Quenching
Quenching refers to any process which decreases the fluorescence intensity of a given substance.
A variety of processes can result in quenching, such as excited state reactions, energy transfer,
complex-formation and collisional quenching. As a consequence, quenching is often heavily
dependent on pressure and temperature.

Examples of quenchers

Molecular oxygen, iodide ions and acrylamideare common chemical quenchers. The chloride
ion is a well known quencher for quinine fluorescence Quenching poses a problem for non-
instant spectroscopic methods, such as laser-induced fluorescence.
Quenching is made use of in optode sensors; for instance the quenching effect of oxygen on
certain ruthenium complexes allows the measurement of oxygen saturation in solution.
Quenching is the basis for Förster resonance energy transfer (FRET) assays Quenching and
dequenching upon interaction with a specific molecular biological target is the basis for
activatable optical contrast agents for molecular imaging

Flourescence and quantum yeild

Luminescence is the emission of light from any substance, and occurs from electronically excited
states. Depending on the nature of the excited state the luminescence is formally divided into two
categories –florescence and phosphorescence. Important characteristics of a florophore is the
flouorescence lifetime and quantum yield.

Quantum yield

This quantum yield is the number of emitted photons relative to the number of absorbed
photons. The intensity of the emission can be decreased by dierent processes. Theses decreases
in intensity are called quenching and can occur by dffierent mechanisms.

Instrumentation

For fluorescence measurements we need to selectively excite molecules to the desired excited
state, requiring light at a certain wavelength or in a limited wavelength range. For this purpose,
commercial fluorescence spectrometers often use the same kind of monochromator arrangement
that is found in absorption spectrometers (Emitted fluorescence is then again frequency selected
(by a second monochromator) and can be recorded as a function of emission wavelength. In this
way, emission from different excited states can be separated and, most importantly, the excitation
light can be suppressed.

Spectrofluorimeter,) Figure 5: Schematic view of a fluorescence spectrometer (top) and a

simple setup for SternVolmer and lifetime analysis In a commercial spectrometer,
excitation and emission wavelengths are selected by monochromators instead of filters

The fluorescence intensity data for the Stern-Volmer analysis can, on the other hand,

be measured with a much simpler setup as shown in Fig. 1. Here the selection of excitation and
detection wavelength is achieved with different filters, and/or by using a narrow-band light
source, such as an LED. If we modulate the LED light source intensity at high frequency (tens of
kHz), we can also measure the phase shift of a long-lived fluorescence signal (>100 ns) and
determine the lifetime without the Stern-Volmer analysis

mechanisms.

One possibility is that the excited-state flouorophore is deactivated upon contact with some
other molecules in solution. In this case,mechanism of quenching is performed by either
following method

collisional quenching

collisional quenching, the uorophore is returned to the ground state during a diusive encounter
with the quencher. Through this process the molecules are not chemically altered. For collisional
quenching the decrease in intensity is described by the well-known Stern-Volmer equation. The
mechanism of quenching varies with the uorophore-quencher pair. For instance, quenching is
probably due to electron transfer, spin-orbit coupling or intersystem crossing to the triplet state.
Besides the collisional quenching, fluorescence quenching can occur by a variety of other
processes, e.g.

Flourescence quenching

The emission of light from the excited state of a molecule (fluorescence or phosphorescence) can
be quenched by interaction with another molecule.this is called fluorescence
quenchingFluorescence quenching can also take place by the formation at the ground state of a
non-fluorescent complex. there is a difference between quenching and decrease of fluorescence
due to the high state of molecular excitement or chemical changes of FLUOROPHORE (like
oxidation).
The stationary and time-dependent observation of such processes reveals insight into the
deactivation mechanisms of the excited molecule and can be used for monitoring distance and
orientation changes between different parts of biomolecules

dynamic quenching

The dynamic quenching occurs within the fluorescence lifetime of the fluorophore, i.e. during
the lifetime of the excited state. This process is time dependent. When non flourescense complex
absorbs light, it immediately returns to the fundamental state without emitting any photons. This
type of complex is called static quenching

Meaurement of fluorescence quenching And quantum mechanics

1.6 Stern-Volmer Method

 The Stern-Volmer-equation describes the dependence of the fluorescent intensity of a
fluorescent dye on the concentration of an quenching material (Quencher). It was created by the
two physical chemists Otto Stern and Max Volmer in 1919.

We can gain information about the fluorescence lifetime and excited state deactivation by
introducing quenchers (heavy ions or acceptor molecules) and observing the fluorescence
intensity as a function of their concentration. To see how this is possible we build a rate model
for the concentration of molecules in the fluorescing excited state S1 and use the following
notations:

[M] Concentration of the fluorescing molecule in the ground state

[M∗] Concentration of the molecule (donor) in the fluorescing

S1 state [Q] Concentration of the quenching molecule (acceptor in the ground state)

Neglecting the possibility photo chemical reactions, the following processes contribute to a
change of [M∗] (

Photo excitation M → M∗ kabs[M]

Fluorescence M∗ → M kf[M∗]

Non-fluorescent relaxation (intramolecular) M∗ → M (or other excited state) knf[M∗]

Quenching (intermolecular) M∗ + Q → M + Q (or Q∗) kq[M∗][Q]

The fraction of excited molecules at any time is usually very small (unless intense pulsed light
sources are used), so [M] ≈ const. Defining Iabs = kabs[M] we obtain the following differential
equation for the excited state population:

∂[M∗]\ ∂t= Iabs −(kf + knf + kq[Q])[M∗] (2)

Without quencher molecules ([Q] = 0) the equation becomes even simpler:

∂[M∗]\ ∂t= Iabs −(kf + knf)[M∗] (3)

Under continuous irradiation a stationary state is quickly established and the excited state
population [M∗] is constant (∂[M∗]/∂t = 0). An important quantity for the determination of
different reaction rate constants is the fluorescence quantum yield Φ:

Φ = number emitted photons\ number absorbed photons or

Φ = rate of emission\ rate of absorption

In our notation, the rate of absorption is Iabs = kabs[M] and the rate of emission is kf[M∗].
With the help of equations 2 and 3 under stationary conditions (∂[M∗]/∂t = 0), we obtain for the
quantum efficiencies with and without quencher molecules:

ΦQ = kf \kf + knf + kq[Q] (5)

and

Φ0 = kf\ kf + knf (6)

The ratio of the quantum yields is equal to the ratio of the observed emission intensities without
and with the quencher molecules: I0\ IQ = Φ0 \ΦQ = kf + knf + kq[Q]\ kf + knf = 1 + 1 kf +
knf \kq[Q] (7)

Inserting the fluorescence lifetime in the absence of the quencher molecules (equation 1) we
obtain the final result (also known as Stern-Volmer equation):

I0\ IQ = 1 + τfkq[Q] (8)

APPLICATIONS OF QUENCHING FLOURESCENCE

Sphingolipid Metabolism and Cell Signaling,

Principles of Using Fluorescence Quenching to Detect Phase Separation: Behavior of Simple

Binary Lipid Mixtures
Fluorescence quenching (the decrease in intensity induced by a quencher molecule) offers a
powerful approach to detection of sphingolipid phase separation

phase separation studies

Quenching is uniquely suited to phase separation studies because it can detect relatively small
domains. In addition, unlike other spectroscopic assays, it does not require that the two phases
have different physical properties. Only a difference in lipid composition in each phase is
necessary to detect phase separation. We have found that quenching is useful in detecting both
liquid–crystalline/gel and liquid–crystalline/liquid-ordered phase separation.
Lipid phase separation depends on both lipid composition and temperature

RNA Polymerases and Associated Factors,

We used a neutral quencher, acrylamide, to measure the accessibility of tryptophan residues in
RNA polymerase to get an idea of changes in tertiary structure. We probed the accessibility of
the Tb–GTP fluorophore bound at the active site of the enzyme in a similar manner. Quenching
data were analyzed according to Stern–Volmer

Fluorescence quenching by heavy atoms such as iodide can be used to determine how exposed a
fluorescently tagged residue is to the solvent.

Measuring binding affinity between ligands and proteins.

Fluorescence quenching is an important technique for measuring binding affinity between

ligands and proteins. Fluorescence quenching is the decrease in the quantum yield of
fluorescence from a fluorophore, induced by a variety of molecular interactions with quencher
molecule
Medical Applications
Quenching is a really good visible physical phenomenon, that's why it is used as an indicator for
molecular processes.

indicator for DNA hybridization

If DNA strand is hybridizing with an opposite strand it gets quite linear and stiff. Fluorphore and
Quencher are attached to the ends. If the bases are paring correctly the Fluorphore and Quencher
disconnect and the quenching stops.
Indicator for Potassium-ions
The ends of short DNA fragments (telomer sequence), connected with a fluorescent dye and
quencher through a covalent bond, are divided in a solution. The dye is producing light. If the
there are any potassium-ions present, the DNA fragment is wrapping around the potassium-ion
and the endings are touching each other. The fluorescence is quenched (lightning stops).