Fluorescence Quenching
Fluorescence Resonance Energy
Transfer
04.10.2012
What happens with the
absorbed energy?
Internal conversion
(heat)
Fluorescence quenching
kic
kQ
kis
„intersystem crossing”
S→T
ENERGY
c
kf
kFRET
FRET
Fluorescence (ns)
Phosphorescence (ms)
Radiative (with light) and non-radiative (without light) transitions!
Rate constant
• Term (phrase) coming from reaction kinetics.
• a constant that correlates the speed (rate) of reaction to the actual concentration of a
substance (cA).
v=−
dc A
= kr c
r
A
dt
kr – rate constant (factor of proportionality)
cA – the actual concentration of the substance A (M)
r – the order of the reaction (the number of components whose concentration can affect the rate of the reaction)
t – time (s)
What happens with the
absorbed energy?
Internal conversion
(heat)
Fluorescence quenching
kic
kQ
kis
„intersystem crossing”
S→T
ENERGY
c
kf
kFRET
FRET
Fluorescence (ns)
Phosphorescence (ms)
Radiative (with light) and non-radiative (without light) transitions!
Fluorescence quenching
• Quenching: any process which can decrease the fluorescence
intensity of a given substance (fluorophore).
• Quencher: a non-fluorescent molecule that can absorb the energy.
• Competition between the fluorescence emission (kf) and the nonradiative transitions (kQ+kic+kisc).
→ decreased fluorescence emission!
Types of quenchers
Neutral (non-polar, uncharged) quenchers: Acrylamide, nitroxide (NO),
oxygen
studying structural differences („space limited”).
Polar (charged) quenchers: Iodide-ion (I-), Caesium-ion (Cs+), Cobalt-ion (Co+)
monitoring the electrostatic differences (charge distribution).
Types of fluorescence quenching (1)
STATIC QUENCHING
slope = KSV
temperature ↑
F0 / F
• Formation
of
dark
(nonfluorescent)
ground
state
complexes.
→ The number of the excited
fluorophores is decreasing → the
fluorescence
intensity
is
decreasing.
τ0 / τ
1
• The
increased
temperature
decrease its effectivity.
• The fluorescence lifetime is not
affected.
0
Q
Stern-Volmer equation
F0 / F = 1+KSV[Q]
KSV: Stern-Volmer quenching constant
Types of fluorescence quenching (2)
• Collision between the fluorophore
and the quencher molecule → the
number of the excited fluorophores
will not change.
• The fluorescence intensity and the
the fluorescence lifetime will
decrease together.
• Diffusion controlled processes can
affect it.
F0 / F and τ0 / τ
DYNAMIC QUENCHING
temperature ↑
slope = KD = kqτ0
1
0
Q
Stern-Volmer equation
F0 / F = τ0 / τ = 1+KD[Q] = 1+ kqτ0[Q]
KD:Stern-Volmer quenching constant
kq: bimolecular rate constant
Interpreting the results
• The Stern-Volmer constant (KSV) can inform about the efficiency of the
quencing = accessibility of the fluorophore.
• bimolecular rate constant (kq)
- information about the accessibility.
- Information about the dynamics of the quenching process.
kq ~ 1 x 1010 M-1s-1 diffusion controlled process.
Quenching of the tryptophan fluorescence
in the presence of Acrylamide
F-actin
G-actin
Quenching of the tryptophan fluorescence
in the presence of Cesium-chloride
F-actin
G-actin
Fluorescence Resonance Energy
Transfer
• Perrin suggested that energy could be transferred over distances longer
than the molecular diameters.
• Theodor Förster developed the theoretical basis of FRET (1946).
• Förster type energy transfer: energy transfer between a donor and an
acceptor molecule through dipole-dipole interactions.
Radiationless: photons are not involved.
• Fluorescence Resonance Energy Transfer (FRET): energy transfer
between fluorophores.
Dipole
• Apolar(non-polar) molecule: uniform charge distribution.
• Polar molecule: non-uniform charge distribution (the
positively and negatively charged parts are separated).
→ Dipole-molecule: polar molecule with two poles.
+
FRET
FRET is a special type of quenching of the fluorescence which can decrease the
fluorescence intensity of a given fluorophore (donor).
hν
hν
-
D
hν
E ~ kt ~ 1/R6
+
A
+
R
Donor: the source of the
fluorescence.
Acceptor: a fluorophore that can
absorb the energy accumulated in
the donor molecule.
Conditions has to be fulfilled
• Fluorescent donor and acceptor molecule.
• The distance (R) between the donor and acceptor molecule 2-10 nm.!
• Overlap between the donor’s emission spectrum and the acceptor’s
absorption spectrum.
How to determine the efficiency
of FRET
• „steady-state” measurements
E = 1 – (FDA / FD)
• time-dependent measurements
(fluorescence lifetime)
E = 1 – (τDA / τD)
Distance dependence of FRET
Förster distance in energy resonance transfer
6
0
R
E= 6
6
R0 + R
distance between the fluorophores
Distance dependence of FRET
1
0.8
E
0.6
0.4
E=
R0
E=
0.2
6
R0
2
2
R0 + R
!!!!!!!
2
6
R0 + R 6
0
0
2
4
6
8
10
R (nm)
12
14
16
18
20
Förster distance – R0 (1)
The distance between the donor and the acceptor molecule where the
measured transfer efficiency is 50 %.
1.1
1
0.9
0.8
0.7
E
0.6
0.5
0.4
0.3
0.2
0.1
0
0
R0
Förster distance – R0 (2)
It is possible to calculate it.
R0 = 0.211 • [η-4 Q0 k2 J(λ)]1/6
(the result is in Å (10-10m))
k2 (k-squre) = orientation factor (the relationship between the donor’s
emission vector and the acceptor absorption vector).
η = refractive index (1.33-1.6)
Q0 = quantum efficieny of the donor in the absence of the acceptor
J(l) = overlap integral (overlapping area between the donor’s emission
spectrum and the acceptor’s absorption spectrum)
Distance dependence of FRET
6
0
R
E = 6
6
R0 + R ?
DISTANCE MEASUREMENT !
Distance dependence of FRET
6
0
R
E = 6
6
R0 + R ?
Example:
E = 70%
R0 = 5 nm
R=?
x=
6
R0
6
−R
0
E
6
6
x=
6
5 −5
0.7
6
= 4.3 nm
FRET
• Molecular ruler on the nanometric scale (10-9m).
• very sensitive!
• Application:
– Studying the interactions between molecules.
–Studying structural changes within molecules.
• The end!