Lecture 29

Introduction to
Fluorescence
Spectroscopy
 Introduction
When a molecule absorbs light, an electron is promoted to a higher excited state (generally a
singlet state, but may also be a triplet state). The excited state can get depopulated in several
ways.
The molecule can lose its energy non
radiatively by giving its energy to
another absorbing species in its
immediate vicinity (energy transfer) or
by collisions with other species in the
medium.
If an excited state triplet overlaps with
the exited state singlet, the molecule can
cross over into this triplet state. This is
known as inter system crossing. If the
molecule then returns to the ground state
singlet (T1 S0) by emitting light, the
process is known as phosphorescence.
The molecule can partially dissipate its energy by undergoing conformational changes
and relaxed to the lowest vibrational level of the excited state in a process called
vibrational relaxation. If the molecule is rigid and cannot vibrationally relax to the
ground state, it then returns to the ground state (S1 S0) by emitting light, the process is
known as fluorescence.
 Jablonski diagram

Emission from T1 is called phosphorescence

S2
Internal conversion
Inter-system
S1 crossing
T1

Fluorescence Phosphorescence

S0
 The Stokes Shift
The energy of emission is typically less
than that of absorption. Fluorescence
typically occurs at lower energies or longer
wavelength.
 Characteristic of a fluorescence
spectra
Kashas Rule: The same fluorescence
emission spectrum is generally observed
irrespective of excitation wavelength. This
happens since the internal conversion is
rapid.
 Some important facts
Upon return to the ground state the fluorophore
can return to any of the ground state vibrational
level.
The spacing of vibrational energy levels of the
excited states is similar to that of the ground
state.
The consequence of above two is that the
emission spectrum is typically a mirror image of
the absorption spectrum of the S0 to S1
transition.
 Fluorescence life times and
quantum yield
Quantum yield is the The lifetime of the
ratio of the number of excited state is
photons emitted to defined by the
the number absorbed. average time the
molecule spends in
the excited state prior
Q
k nr to the return to the
ground state.
= the emissive rate of
1
fluorophore.
knr= rate of non-radiative k nr
decay
 Fluorescence life time and
quantum yield
The lifetime of the Fluorescence lifetimes
fluorophore in the are near 10 ns.
absence of radiative Scintillators have large
process is called the value. Hence they have
intrinsic or natural life large Q and lifetime.
time. The fluorescence
emission of aromatic
substances containing
1 nitro group are generally
n
weak due to large knr
value.
 Absorption versus Emission
spectra
Absorption is an instantaneous
event. It occurs so fast that
there is no time for molecular
motion during the absorption
process. Thus absorption
spectra are not sensitive to
molecular dynamics and can
provide information on average
solvent shell adjacent to the
chromophore.
In contrast to absorption,
emission occurs over a longer
period of time.
The length of time fluorescent
molecules remain in excited
state provides an opportunity
for interactions with other
molecule in solution like
oxygen.
Other example of dynamic
processes in solution involve
fluorophore-solvent
interactions and rotational
diffusion.
 In terms of energy level
The emitted light is always of lower energy (longer wavelength). This is known as the
Stokes shift.

The quantum yield (Q) of a fluorescence

phenomena is given by:

Q = Number of photons emitted

Number of photons absorbed

In a given solvent, the quantum yield of a particular fluorophore will be fixed.

Because of this, every fluorophore will have a characteristic fluorescence spectrum
 Scope of quenching and energy loss during
fluorescence
.
Energy may be lost in vibrational transition, collision with the solvent, heat
transfer etc. .Only a part of the light absorbed is emitted. Its because of this that
the quantum yield in most practical cases is not equal to one.

Quenching of fluorescence may also occur due to the presence of some foreign
molecule in the solution which is acting as a quencher, or due to some structural
rearrangement in the molecule (say protein), which drives the fluorophore to a
conformation where it is in vicinity of a quencher (any amino acid residue or
disulphide bond).

Fluoresence intensity may also decrease due to the transfer of the emitted
energy to some other chromophore, which absorbs at that energy. This
phenomena is called FRET. However, FRET and quenching should not be
treated synonymously
 Experimental Set Up

I0 I
excitation excitation
Excitation
Monochromator Cuvette with
Light sample
source

Emission
Emission spectrum Excitation wavelength Monochromator
is kept constant and fluorescence intensity emission
measured as function of wavelength, i.e.
spectrum of emitted light is determined.
Excitation spectrum Fluorescence
intensity at a particular fixed wavelength is
measured as a function of the excitation Detector
wavelength.
 The effect of solvent on the
fluorescence spectra
The effect of solvent and environment may be
due to several factor
Solvent polarity and viscosity
Rate of solvent relaxation
Probe conformational changes
Rigidity of the local environment
Internal charge transfer
Proton transfer and excited state reaction
Probe-Probe interaction.
 Effect of solvent
Typically, the fluorophore has a larger dipole moment in the
excited state than the ground state. Solvent shifts the emission to
lower energy due to stabilization of the excited state by the polar
solvent molecule. As the solvent polarity is increased , this
effect becomes larger.
S2
Internal conversion
S1
Less polar solvent

More polar
solvent

S0
 Lecture 30

Quenching and FRET

 Quenching
Fluorescence quenching refers to any
process that decreases the fluorescence
intensity of a sample.
A variety of molecular association can
result in quenching. These include excited-
state reactions, molecular
rearrangements, energy transfer, ground-
state complex formation, and collisional
quenching.
 Quenchers
A wide variety of substances act as
quenchers of fluorescence. Quenching by
oxygen is due to its paramagnetic nature
causes the fluorophore to undergo
intersystem crossing to the triplet state.
 Stern-Volmer Plot

F0
F

Q
Static quenching
KS
F Q
F Q

F 0 F F Q

KS
F 0 F
F Q

K S Q
F 0 1
F

F 0 1 K s Q
F
Static quenching
Combined static and dynamic
quenching
 Frsters Resonance Energy Transfer (FRET)

Excite Fluorescence emission from the donor

(D) is absorbed by the acceptor (A).
The emission spectrum of donor and the
A Emission absorption spectrum of the acceptor must
D have a spectral overlap.
FRET is a non-radiative process. The
FRET efficiency is dependent on the
Emission-absorption distance between the donor and acceptor.

Donor emission spectra

Acceptor absorbance spectra

Spectral overlap

Wavelength (nm)
 FRET as a spectroscopic ruler
Unique locations of the donor and the acceptor in the protein molecule allows one to monitor
the conformation of the protein with respect to the position of the donor and acceptor, because
FRET is dependent on the distance between D and A. Conformational change in the protein
will lead to an increase or decrease in the FRET efficiency, along with the transfer rate. This
can in turn give an idea of the distance between D and A- Spectroscopic ruler. It is ideal
for measuring distance ranging from 10 to 80, relevant to biological system.

R06
The FRET efficiency is given by E 6 6
R0 r
6
1 R0
kT
The rate of transfer of energy is given by
D r
r is the distance between D and A, D is the decay time of D in absence of A, R0 is the Forsters
distance, which can be calculated from the absorption and emission spectra. E can be
calculated from the emission spectra.
FDA
E
FD
FDA is the fluorescence in presence of acceptor, while FD is the fluorescene in absence of
acceptor.
When r=R0, the transfer efficiency is 50%. The rate of transfer is equal to 1/D, when r=R0.
The rate of energy transfer is dependent on r-6.
 FRET Spectrum

One of the reason for the low fluorescence of Phe in protein is its energy transfer to
Tyr and Trp.
Energy transfer is also one of the reason why Tyr fluorescence is not observed in
proteins which contain both Try and Trp. That is, energy transfer can occur from Tyr
to Trp in the compact native state.
Life-time measurement
 Life time
Life time measurements can reveal how each of flurophore is
affected by interaction if there are more than one fluorophore.

Can distinguish between static and dynamic quenching.

Resonance energy transfer can also be best studied using life-

time.

Fluorescence life times are typically independent of probe

concentration, hence often used for cellular imaging.
Life time is Time for intensity to drop by 1/e
Time-correlated single photon
counting
Time domain lifetime
Frequency domain Life-time