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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