Lecture 29

Introduction to Fluorescence Spectroscopy

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.

Introduction

• 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

Internal conversion Inter-system

crossing

Phosphorescence Fluorescence

S0

S1

S2

T1

Emission from T1 is called phosphorescence

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

• Kasha’s 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 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.

nrkQ

Γ= the emissive rate of

fluorophore.

knr= rate of non-radiative

decay

nrk

1

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.

• 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.

1n

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

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

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

Light

source

Excitation

Monochromator Cuvette with

sample

Detector

I0 I

Emission

Monochromator

λexcitation λexcitation

λemission

Experimental Set Up

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.

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

Internal conversion

S0

S1

S2

Less polar solvent

More polar

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.

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

F

F0

Q

1

Static quenching

QKF

F

F

FQK

QF

FFK

QFFF

QF

QFK

s

S

S

S

1

1

0

0

0

0

Static quenching

Combined static and dynamic quenching

Förster’s Resonance Energy Transfer (FRET)

D A

Excite

Emission

Emission-absorption

•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.

Spectral overlap

Donor emission spectra

Acceptor absorbance spectra

Wavelength (nm)

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.

The FRET efficiency is given by 66

0

6

0

rR

RE

The rate of transfer of energy is given by

6

01

r

Rk

D

T 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.

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.

D

DA

F

FE

FDA is the fluorescence in presence of acceptor, while FD is the fluorescene in absence of

acceptor.

FRET as a spectroscopic ruler

•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.

FRET Spectrum

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