Fluorescence SpectroscopyFluorescence Spectroscopy

Part I. Background

Perrin-Jablonski Perrin-Jablonski diagramdiagram

S is singlet and T is triplet.

The S0 state is the ground state and the subscript numbers identify individual states.

n→*→*n→*→*→

*

Energy level of MOEnergy level of MO

S0

Singlet & TripletSinglet & Triplet

Characteristics of Excited StatesCharacteristics of Excited States

Energy

Lifetime

Quantum Yield

Polarization

Stokes shiftStokes shiftThe Stokes shift is the gap between the maximum of the first absorption band and the maximum of the fluorescence spectrumloss of vibrational energy in the excited state as heat by collision with solvent

heat

ExampleExample: 7-amino-4-methylcoumarin (AMC)

ExampleExample

Example Example fluorophores

fluorescein

ethidium bromide

bound to DNA.

LifetimeLifetime

LifetimeLifetime

Excited states decay exponentially with time

– I = I0e-t/

• I0 is the initial intensity at time zero,

• I is the intensity at some later time t

• is the lifetime of the excited state.

• kF = 1/, where kF is the rate constant for fluorescence.

Quantum YieldQuantum Yield = F

• F = number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited state

• F = ratio of photons emitted to photons absorbed

•Quantum yield is the ratio of photons emitted to photons absorbed by the system:

Quantum YieldQuantum Yield

Quantum Yield & Structure rigidityQuantum Yield & Structure rigidity

Polarization• Molecule of interest is randomly oriented in a rigid matrix

(organic solvent at low temperature or room temperature polymer). And plane polarized light is used as the excitation source.

• Degree of polarization is defined as P

I|| and Iare the intensities of the observed parallel and perpendicular components,

is the angle between thee mission and absorption transition moments.

If is 0° than P = +1/2,

and if is 90° than P = -1/3.

• Steady-state measurements: , I

• Time-Resolved measurements:

Experimental MeasurementsExperimental Measurements

InstrumentsInstruments

Inner Filter Effect Inner Filter Effect

• At low concentration the emission of light is uniform from the front to the back of sample cuvette.

• At high concentration more light is emitted from the front than theback.

• Since emitted light only from the middle of the cuvette is detected the concentration must be low to assure accurate F measurements.

Inner Filter Effect

If (em) = IAbs (ex). f . f(em)

. K

I0(ex)

emem

em

em

measured intensity of fluorescence at em

absorbed intensity at ex

fluorescence quantum yield

fraction of intensity emitted at thatparticular wavelength

fraction of total fluorescencethat is detected

)(0 101)()( exA

exexabs II

If A0 exexexabs IAI 0..303.2

KIAKII fexex

em

fexabsf .).().(.303.2.).( 0

standard

sample

standard

sample

standard

sample

.f

f

ex

ex

em

f

em

f

A

A

I

I

If we measure the sample and a standard under thesame experimental conditions, keeping ex constant:

Important : the index of refraction of the two solvents(sample and standard) must be the same

Standards:

Quinine sulfate in H2SO4 1N: f =0.55

Fluorescein in NaOH 0.1N: f =0.93

Measurement of fluorescence quantum yieldsMeasurement of fluorescence quantum yields

The TCSPC measurement relies on the concept that the probability distribution for emission of a single photon after an excitation yields the actual intensity against time distribution of all the photons emitted as a result of the excitation. By sampling the single photon emission after a large number of excitation flashes, the experiment constructs this probability distribution.

Time correlated single photon counting:

#eve

nts

.

.

.

.

t (nsec)

diff

eren

t ex

cita

tion

flash

esStart PMT

Stop PMTsample

exc. monochromator

em

iss

ion

mo

no

ch

rom

ato

r

pulsed source

t

Measurement of fluorescence lifetimesMeasurement of fluorescence lifetimes

Lifetimens

Absorption Fluorescence

Wavelength nm

Absorptivity Wavelength

nmQuantum

Tryptophan 2.6 280 5,600 348 0.20

Tyrosine 3.6 274 1,400 303 0.14

Phenylalanine 6.4 257 200 282 0.04

Intrinsic Fluorescence of Proteins and PeptidesIntrinsic Fluorescence of Proteins and Peptides

•TryptophanTryptophan, the dominant intrinsic fluorophore, is generally present at about 1mol% in proteins. A protein may possess just one or a few Trp residues, which facilitates interpretation of the spectral data.

•TryptophanTryptophan is very sensitive to its local environment. It is possible to see changes in emission spectra in response to conformational changes, subunit association, substrate binding, denaturation, and anything that affects the local environment surronding the indole ring. Also, Trp appears to be uniquely sensitive to collisional quenching, either by externally added quenchers, or by nearby groups in the protein.

•Tryptophan Tryptophan fluorescence can be selectively excited at 295-305 nm. (to avoid excitation of Tyr)

TryptophanTryptophan

I II III

IVV

ExampleExample: Tyrosine and its derivatives

I

III

III

III

IV

IV

II

V

V

Emission spectra of Pseudomonas fluorescens azurin Pfl.For 275-nm excitation, a peak is observed due to the tyrosine residue(s)

• The position and structure of the fluorescence suggests that the indole residue is located in a completely nonpolar region of the protein. These results agree with X-ray studies, which show that the indole group is located in the hydrophobic core of the protein.• In the presence of a denaturing agent, the TrpP emission loses its structure and shifts to 351nm, characteristic of a fully exposed Trp residue. Changes in emission spectra can be used to follow protein unfolding

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Resolution of the contributions of individual tryptophan residues in multi-tryptophan proteins.

I(,t)=i()exp(-t/i)i

1=2ns, 2= 5ns

0

20

40

60

80

100

120

500 550 600 650 700 750

1=2ns

2=5ns

t (ns)

Flu

ore

sce

nce

inte

nsi

ty (

A.U

.)F

luo

resc

en

ce in

ten

sity

(A

.U.)

wavelength (nm)

em

Example Time-resolved protein fluorescenceExample Time-resolved protein fluorescence

Isolated from the Pacific jellyfish Aequorea victoria and now plays central roles in biochemistry and cell biology due to its widespread use as an in vivo reporter of gene expression, cell lineage, protein protein interactions and protein trafficking One of the most important attributes of GFP which makes it so useful in the life sciences is that the luminescent chromophore is formed in vivo, and can thus generate a labeled cellular macromolecule without the difficulties of labeling with exogenous agents.

Green fluorescent protein (abbreviated GFPGreen fluorescent protein (abbreviated GFP

The structure of GFP : eleven-strand beta-barrel wrapped around a central alpha-helix core. This central core contains the chromophore which is spontaneously formed from a chemical reaction involving residues Ser 65, Tyr 66, and Gly 67 (SYG)There is cyclization of the polypeptide backbone between Ser 65 and Gly 67 to form a 5-membered ring, followed by oxidation of Tyr 66.The high quantum yield of GFP fluorescence probably arises from the nearly complete protection of the fluorophore from quenching water or oxygen molecules by burial within the beta-barrel.

Ribbon diagram of the Green Fluorescent Protein (GFP) drawn from the wild-type crystal structure. The buried chromophore, which is responsible for GFP's luminescence, is shown in full atomic detail.

Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum. For wild type GFP, exciting the protein at 395 nm leads to rapid quenching of the fluorescence with an increase in the 475 nm excitation band. This photoisomerization effect is prominent with irradiation of GFP by UV light. In a wide range of pH, increasing pH leads to a reduction in fluorescence by 395 nm excitation and an increased sensitivity to 475 nm excitation.

Melittin GIGAVLKVLT TGLPALISWI KRKRQQX

Example CarboxyfluorescenceCarboxyfluorescence

Biochemical Education 28 (2000) 171

~173

Example CarboxyfluorescencCarboxyfluorescencee

Quenching EffectQuenching Effect

Example CarboxyfluorescenceCarboxyfluorescence

pH EffectpH Effect