Slide 1

Report
Big Question: We can see rafts in Model Membranes (GUVs or
Supported Lipid Bilayers, LM), but how to study in cells? Do rafts
really exist in cells? Are they static large structures? Are they small
transient structures?
FRET and FRET based Microscopy Techniques
4 basic rules of fluorescence for overview presentation:
•The Frank-Condon Principle: the nuclei are stationary during the electronic
transitions, and so excitation occurs to vibrationally excited electronic states.
•Emission occurs from the lowest vibrational level of the lowest excited singlet
state because relaxation from the excited vibrational energy levels is faster than
emission
•The Stokes Shift: emission is always of lower energy than absorption due to
nuclear relaxation in the excited state
•The mirror image rule: emission spectra are mirror images of the lowest energy
absorption
Fluorescence
Jablonski Diagram
Stokes shift is the difference (in
wavelength or frequency units) between
positions of the band maxima of the
absorption and luminescence spectra of the
same electronic transition.
When a molecule or atom absorbs light, it
enters an excited electronic state. The
Stokes shift occurs because the molecule
loses a small amount of the absorbed
energy before re-releasing the rest of the
energy as luminescence. This energy is
often lost as thermal energy.
E = hn = hc/l
Frank-Condon Principle and Leonard-Jones Potential
Factors Governing Fluorescence Intensity
1) Internal conversion – non radiative loss via collisions with solvent or
dissipation through internal vibrations. In general, this mechanism is
dependent upon temperature. As T increases, the rate of internal
conversion increases and as a result fluorescence intensity will
decrease.
2) Quenching – interaction with solute molecules capable of quenching
excited state. (can be various mechanisms)
O2 and I- are examples of effective quenchers
3) Intersystem Crossing to Triplet State.
Quantum Yield : number of photons emitted/number of photons absorbed.
Quenching
Common Fluorescence Applications in Biophysics:
Tryptophan Fluorescence – Protein Folding/Binding Isotherms
Fluorescence Quenching - Protein Structure and Dynamics
Fluorescence Anisotropy – Binding
Fluorescence Resonance Energy Transfer – Binding, Distances, Conformations
Common Fluorescent Probes
Sensitivity to Local Environment:
Fluorescence can be used to probe local environment because of the
relatively long lived singlet excited state.
10-9 to 10-8 sec, various molecular processes can occur
•Protonation/deprotonation
•Solvent cage reorganization
•Local conformational changes
•Translations/rotations
example: (a) intensity and wavelength of fluorescence
can change upon going from an aqueous to non-polar
environment. This is useful for monitoring
conformational changes or membrane binding.
(b) Accesibility of quenchers, location on surface,
interior, bilayer etc.
FRET: Fluorescence Resonance Energy Transfer
•
•
•
•
Sensitive to interactions from 10 to 100Å
Increase acceptor sensitivity
Quenches donor fluorescence
Decreases donor lifetime
Overlap Integral
Transition Dipoles
FRET: Fluorescence Resonance Energy Transfer
Rate:
kt=d-1(Ro/R)6
kt =rate of rxn
d =lifetime of donor
R=distance between
fluorophores
Ro =Förster distance
Förster distance
Ro=(2*J(l)*n-4*Q)1/6*9.7*102
J(l) =overlap integral
2 =transition dipoles of
fluorophores
n=refractive index of
medium
Q=quantum yield
% transfer = Efficiency (E):
Quenching:
E= 1-(I/Io)
Energy Transfer:
E=(ad(l1)/da(l1))*[(Iad(l2)/Ia(l2))-1]
I=intensity with FRET
Io=intenstiy without FRET
ad=absorption of accepter (with
donor)
da=absorption of donor (with
acceptor)
Fluorescence Anisotropy
Plane polarized light to exite, detect linearly
polarized light.
Any motion that occurs on the time scale of
the lifetime of the excited state, can modulate
the polarization. Hence, this technique is
used to measure size, shape, binding and
conformational dynamics
FRET with Anisotropy:
Fret Apps to Bilayers
GM1 toxin
GPI anchored proteins
GFP
Homo versus Hetero Fret
FRET fluorescence resonance
energy transfer

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