1. Special Applications in Fluorescence Spectroscopy Miklós Nyitrai; 2007 March 14

2. Fl. reminder Aleksander Jablonski (1898-1980) Polish physicist The Jablonski scheme

3. Definitions Lumin.-fluor.-phosphor. Spectra Fluorescence lifetime Fluorescence quantum yield Anisotropy

4. 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

5. De-excitation or decay processes

6. 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

7. decay excitation How to understand the rate constants? k1k1 k 2 (+k 3 k 4 k 5 k 6... k i ) The probability for each decay process can be calculated as: k i / k sum

8. decay excitation The interpretation of fluorescence? k0k0 kfkf Fluorescence intensity k f / (k 0 + k f ) (  = N emitt / N abs )

9. excitation What happens if a new decay process is involved? k0k0 kfkf Decrease of the intensity! E.g. fluorescence quenching! k f / (k 0 + k f + k n ) knkn

10. excitation How to interpret the fluorescence lifetime? Reminder: decay curve! k0k0 kfkf The lifetime decreases due to the new decay process! k0k0 kfkf knkn knkn E.g. fluorescence quenching!

11. 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!

12. 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.

13. Static quenching Dark complexes are formed between the ground state fluorophore and the quencher.  The complex is formed at the moment of excitation.

14. F0F0 Quencher concentration How to measure fluorescence quenching? The fluorescence intensity is measured at different quencher concentrations. F1F1 F2F2

15. The Stern-Volmer equation F 0 / F = τ 0 / τ = 1+K SV [Q] = 1+ k q τ 0 [Q] How to interpret the quenching experiments? Fluorescence intensity (lifetime) vs. quencher concentration. Quencher concentration Fluorescence intensity F 0 / F Slope: K SV 1 Quencher concentration

16. Experimentally determined: the Stern-Volmer constant (K SV ). K SV = k q  0 The solvent accessibility of the fluorophore is characterised by the bimolecular quenching constant (k q ). k q = 1 x 10 10 M -1 s -1  diffusion controlled k q < 1 x 10 10 M -1 s -1  steric shielding of the fluorophore The meaning of the results

17. How to separate dynamic and static quenching? F 0 / F = τ 0 / τ = 1+K SV [Q] NOT sensitive to static quenching! What is different in their effect?

18. 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

19. An example: The quenching of tryptophane fluorescence in actin monomers and filaments.

20. Actin monomer Subdomain 1 Subdomain 4 Subdomain 3 Subdomain 2

21. The results with acrylamide monomer filament

22. Results with cesium-chloride monomer filament

23. A special fluorescence quenching: Fluorescence Resonance Energy Transfer (FRET)

24. 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

25. 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.

26. 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.

27. 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)!

28. What is the spectral overlap? wavelength (nm) Absorption or fluorescence emission

29. FRET The relaxation of the donor through the acceptor molecule! + - A + - D E k t ~ 1/R 6 hνDhνD hνAhνA hνGhνG R FRET Jablonski- scheme

30. The FRET Efficiency E = 1 – (F DA / F D ) where F DA : donor intensity in the presence of acceptor; F D : donor intensity in the absence of acceptor. Can also be determined by fluorescence lifetimes! E = 1 – (τ DA / τ D )

31. The Förster critical distance: R 0 The Förster critical distance is the distance at which the transfer efficiency is 0.5 (50 %). Typical values: DonorAcceptorR o (Å) FluoresceinTetramethylrhodamine55 IAEDANSFluorescein46 EDANSDabcyl33 Fluorescein 44 BODIPY FL 57 FluoresceinQSY 7 and QSY 9 dyes61

32. The distance dependence of FRET FRET is a spectroscopic ruler, which can be used to determine molecular distances!

33. The distance dependence of FRET The donor and acceptor distance in R 0 units FRET efficiency

34. Typical applications of FRET distance measurements! → To study whether there is an interaction between biological objects → Structural changes within a macromolecule FRET

35. How to do an experiment? 1.Find and characterise appropriate fluorophore pairs. 2.Measure the fluorescence intensities. 3.Calculate FRET efficiency. 4.Calculate distance.

36. 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 ?

37. The binding of ANN to myosin head The potential locations for ANN (donor) Acceptor labelling sites.

38. The ANN binds to Ser-181! The binding of ANN to myosin head