Special Applications in Fluorescence Spectroscopy

Miklós Nyitrai; 2007 March 14

Fl. reminder

Aleksander Jablonski (1898-1980)

Polish physicist

The Jablonski scheme

DefinitionsLumin.-fluor.-phosphor.

Spectra

Fluorescence lifetime

Fluorescence quantum yield

Anisotropy

The Interactions of photons and molecules

• Photons and Molecules light scattering

absorption

• Energy → heat (internal conversion)→ Fluorescence (ns)→ Phosphorescence (ms)

→ Fluorescence quenching→ Fluorescence Resonance Energy Transfer

De-excitation or decay processes

excitation

decay

How to model the decay processes?

In the ‘Steady-state’ case the incoming and outgoing amount is the same in every time interval.

A virtual tank

decay

excitation

How to understand the rate constants?

k1 k2 (+k3 k4 k5 k6... ki)

The probability for each decay process can be calculated as: ki / ksum

decay

excitation

The interpretation of fluorescence?

k0 kf

Fluorescence intensity

kf / (k0 + kf )

( = Nemitt / Nabs )

excitation

What happens if a new decay process is involved?

k0 kf

Decrease of the intensity!

E.g. fluorescence quenching!

kf / (k0 + kf + kn )

kn

excitation

How to interpret the fluorescence lifetime?

Reminder: decay curve!

k0 kf

The lifetime decreases due to the new decay process!

k0 kfkn kn

E.g. fluorescence quenching!

What is fluorescence quenching?

• The decrease of the fluorescence intensity by molecules able to interact with the fluorophores.

• Quencher: the molecule responsible for quenching!

• The quenching process competes with the fluorescence decay decrease in fluorescence intensity!

Types of fluorescence quenching

Dynamic quenching

• Due to the collision between the excited state fluorophore and another molecule some of the fluorophores are de-excited by the quencher.

•Diffusion controlled!

•If the probability of the quenching is close to 1 in a collision: strong quencher.

Static quenching

• Dark complexes are formed between the ground state fluorophore and the quencher. The complex is formed at the moment of excitation.

F0

Quencher concentration

How to measure fluorescence quenching?The fluorescence intensity is measured at different quencher concentrations.

F1F2

TheThe Stern-Volmer Stern-Volmer equationequation

F0 / F = τ0 / τ = 1+KSV[Q] = 1+ kqτ0[Q]

How to interpret the quenching experiments?

Fluorescence intensity (lifetime) vs. quencher concentration.

Quencher concentration

Flu

ores

cenc

e in

tens

ity

F0 /

F

Slope: KSV

1

Quencher concentration

Experimentally determined: the Stern-Volmer constant (KSV).

KSV = kq 0

The solvent accessibility of the fluorophore is characterised by the bimolecular quenching constant (kq).

kq = 1 x 1010 M-1s-1 diffusion controlled

kq < 1 x 1010 M-1s-1 steric shielding of the fluorophore

The meaning of the results

How to separate dynamic and static quenching?

F0 / F = τ0 / τ = 1+KSV[Q]

NOT sensitive to static quenching!

What is different in their effect?

• Neutral quenchers: acrylamide, nitroxids characterisation of steric shielding of the fluorophore

• Charged quenchers: iodide, cesium, cobalt characterisation of electrostatic properties around the fluorophores

Types of quenchers

An example:

The quenching of tryptophane fluorescence in actin monomers and

filaments.

Actin monomer

Subdomain 1

Subdomain 4

Subdomain 3

Subdomain 2

The results with acrylamide

monomer

filament

Results with cesium-chloride

monomer

filament

A special fluorescence quenching:

Fluorescence Resonance Energy Transfer

(FRET)

The Interactions of photons and molecules

• Photons and Molecules light scattering

absorption

• Energy → heat (internal conversion)→ Fluorescence (ns)→ Phosphorescence (ms)

→ Fluorescence quenching→ Fluorescence Resonance Energy Transfer

Fluorescence Resonance Energy Transfer

(FRET) - Theodor Förster, 1948

Non-radiative dipol-dipol interaction between a fluorescence donor and an acceptor. The donor gives the excited state energy to the acceptor.

What is the dipol-dipol interaction?

Apolar molecule: homogenous charge distribution Polar molecule: heterogeneous charge distribution,

where the center of positive and negative charges is not the same.

→ Dipol-molecule: a polar molecule with two poles.

The criteria for FRET

• Fluorescent donor.•The appropriate orientation of the donor and acceptor dipoles.•Overlap between the emission of the donor and the absorption of the acceptor.•Proximity: distance range between 2-10 nm (typically)!

What is the spectral overlap?

wavelength (nm)

Ab

sorp

tion

or

fluor

esce

nce

e

mis

sion

FRETThe relaxation of the donor through the acceptor molecule!

+

-

A

+

-

DE

kt ~ 1/R6

hνD

hνA

hνG

R

FRET Jablonski-scheme

The FRET Efficiency

E = 1 – (FDA / FD)

where

FDA: donor intensity in the presence of acceptor;FD : donor intensity in the absence of acceptor.

Can also be determined by fluorescence lifetimes!

E = 1 – (τDA / τD)

The Förster critical distance: R0

The Förster critical distance is the distance at which the transfer efficiency is 0.5 (50 %).

Typical values:

Donor Acceptor Ro (Å)

Fluorescein Tetramethylrhodamine 55

IAEDANS Fluorescein 46

EDANS Dabcyl 33

Fluorescein Fluorescein 44

BODIPY FL BODIPY FL 57

Fluorescein QSY 7 and QSY 9 dyes 61

The distance dependence of FRET

660

60

RR

RE

FRET is a spectroscopic ruler, which can be used to determine molecular distances!

The distance dependence of FRET

The donor and acceptor distance in R0 units

FR

ET

effi

cien

cy

Typical applications of FRET• distance measurements!

→ To study whether there is an interaction between biological objects→ Structural changes within a macromolecule

FRET

How to do an experiment?

1. Find and characterise appropriate fluorophore pairs.

2. Measure the fluorescence intensities.

3. Calculate FRET efficiency.

4. Calculate distance.

An example for FRET applications:

The binding of 9-Anthroylnitrile (ANN) to myosin head

From previous studies: only 1 of the 12 serins can be labelled with ANN.

? But which one ?

The binding of ANN to myosin head

The potential locations for ANN (donor)

Acceptor labelling sites.

The ANN binds to Ser-181!

The binding of ANN to myosin head