1. 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

2. 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

3. 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. wavelengthfrequency absorptionluminescencespectra 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.luminescence Jablonski Diagram Fluorescence E = h  hc 

5. Frank-Condon Principle and Leonard-Jones Potential

6. 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.

7. Quenching

8. 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

9. Common Fluorescent Probes

11. 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.

12. FRET: Fluorescence Resonance Energy Transfer Sensitive to interactions from 10 to 100Å Increase acceptor sensitivity Quenches donor fluorescence Decreases donor lifetime Overlap Integral

13. Transition Dipoles

14. FRET: Fluorescence Resonance Energy Transfer Quenching: Energy Transfer: Rate: k t =  d -1 (R o /R) 6 k t =rate of rxn  d =lifetime of donor R=distance between fluorophores R o =Förster distance Förster distance R o =(  2 *J( )*n -4 *Q) 1/6 *9.7*10 2 J( ) =overlap integral  2 =transition dipoles of fluorophores n=refractive index of medium Q=quantum yield % transfer = Efficiency (E): Quenching: E= 1-(I/I o ) Energy Transfer: E=(  ad ( 1)/  da ( 1))*[(I ad ( 2)/I a ( 2))-1] I=intensity with FRET I o =intenstiy without FRET  ad =absorption of accepter (with donor)  da =absorption of donor (with acceptor)

16. 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

17. FRET with Anisotropy:

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

20. FRET fluorescence resonance energy transfer