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

wavelength is the difference (in 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 = h n = 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) O 2 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 100 Å Increase acceptor sensitivity Quenches donor fluorescence Decreases donor lifetime

Overlap Integral

Transition Dipoles

FRET: Fluorescence Resonance Energy Transfer

Rate: k

t

=

 d -1

(R

o

/R)

6

Förster distance R

o

=(

 2

*J(

l

)*n

-4

*Q)

1/6

*9.7*10

2 k t =rate of rxn  d =lifetime of donor R=distance between fluorophores R o =Förster distance J( l ) =overlap integral  2 =transition dipoles of fluorophores n=refractive index of medium Q=quantum yield I=intensity with FRET

% transfer = Efficiency (E):

Quenching: E= 1-(I/I o ) Energy Transfer: E=(  ad ( l 1)/  da ( l 1))*[(I ad ( l 2)/I a ( l 2))-1] I o =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:

GM1 toxin GPI anchored proteins GFP Homo versus Hetero Fret Fret Apps to Bilayers

FRET fluorescence resonance energy transfer