luorescence

1. Fluorescence
Spectroscopy

2. Introductio
n
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.

3. Jablonski diagram
Emission from T1 is called phosphorescence
S2
Internal conversion
Inter-system
crossing
S1
T1
Fluorescence Phosphorescence
S0

4. The Stokes Shift
 The energy of emission is typically less
than that of absorption. Fluorescence
typically occurs at lower energies or longer
wavelength.

5. Characteristic of a fluorescence
spectra
 Kasha’s Rule: The same fluorescence
emission spectrum is generally observed
irrespective of excitation wavelength. This
happens since the internal conversion is
rapid.

6. 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.

8. Fluorescence life times and quantum yield
 Quantum yield is the ratio of
the number of photons
emitted to the number
absorbed.
 The lifetime of the excited
state is defined by the
average time the molecule
spends in
the excited state prior to the
return to the ground state.
Q 

  knr
Γ= the emissive
rate of
fluorophore.
k = rate of non-
radiative
nr
decay
 
1
  knr

9. Fluorescence life time and quantum yield
 The lifetime of the
fluorophore in the
absence of radiative
process is called the
intrinsic or natural life
time.
 
1

n
 Fluorescence lifetimes are near
10 ns.
 Scintillators have large Γ value.
Hence they have large Q and
lifetime.
 The fluorescence emission of
aromatic substances containing
nitro group are generally weak
due to large knr value.

11. 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.

12. In terms of energy
level
The emitted light is always of lower energy (longer wavelength). This is known as the
Stoke’s 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

15. 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. It’s 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

16. Experimental Set
Up
Light
source
Excitation
Monochromator
I0
λexcitatio
n
I
λexcitati
on
Cuvette with
sample
Emission spectrum – Excitation wavelength
is kept constant and fluorescence intensity
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
wavelength.
λemission
Emission
Monochromator
Detector

17. 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.

22. Quenching and FRET

23. 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.

24. 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.

26. Stern-Volmer Plot
F0 F
1
Q

27. 0
0
0
F  
F 
FQ
S
Static quenching F  Q
KS 
F
Q
F 0  F 
F  Q
S
K 
K Q

F
F  1
F
F
 1  Ks
Q

28. Static quenching

29. Combined static and dynamic
quenching

31. Förster’s Resonance Energy Transfer
(FRET)
Excite
D
A Emission
•Fluorescence emission from the donor
(D) is absorbed by the acceptor (A).
•The emission spectrum of donor and the
absorption spectrum of the acceptor must
have a spectral overlap.
•FRET is a non-radiative process. The
FRET efficiency is dependent on the
distance between the donor and acceptor.
Emission-absorption
Donor emission spectra
Acceptor absorbance spectra
Spectral overlap
Wavelength (nm)

35. 0

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 80Å, relevant to biological system.
R6
The FRET efficiency is given by E  0
6
k  0
 r 1  R 
The rate of transfer of energy is given by T  
 r 
R6 6
D
r is the distance between D and A, D is the decay time of D in absence of A, R0 is the Forster’s
distance, which can be calculated from the absorption and emission spectra. E can be
calculated from the emission spectra.
E  FDA
F D
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.

36. 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.

37. Life-time measurement

38. 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.

41. Life time is Time for intensity to drop by 1/e

44. Time-correlated single photon
counting

52. Time domain lifetime

53. Frequency domain Life-time

54.  Thanks
 AIPS
 IMRAN KHAN