1. Luminescence (Miklós Nyitrai; 27 th of February, 2007)

2. Energy levels

3. Transitions between levels

4. The Kasha rule

5. Time-scale of the changes

6. Definition of fluorescence and phosphorescence S T S S In the ns range In the > ms range

7. What is fluorescence spectra? Definition: the wavelength dependence of fluorescence emission a.Emission spectra b.Excitation spectra

8. Stokes shift, mirror image

9. Phosphorescence

10. Fluorescence quantum yield Rate constants!

11. Fluorescence lifetime

12. Radiation lifetime How to define fluorescence lifetime? Fluoresc. lifetime

13. How to measure fluorescence: the steady-state case

14. The advantages -great sensitivity and low detection limit -fluorophores are sensitive to the environment

15. How to measure fluorescence lifetime? Time Correlated Single Photon Counting Frequency Domain Principle Two basic common methods:

16. Time Correlated Single Photon Counting

17. After collecting the data make a fit!

18. A practical experiment (TCSPC)

19. Frequency Domain Principle

20. Comparison of the two methods nitrobenzoxadiazol

21. Light sources: lamps and lasers

22. Optical filters

24. Monochromators, polariser, Cuvettes, detectors

25. Classification of types of luminescence

26. The scheme of the reaction

27. Expected to be linear, but…! The inner-filter effect: Reabsorption of emitted fluorescence radiation Concentration dependence of fluorescence

28. Fluorescence probes, dyes: intrinsic fluorophores (definition) Tryptophan, tyrosin, phenilalanine Advantage: no modification of the protein is required.

29. Fluorescence probes, dyes: extrinsic fluorophores - e.g., dansyl, fluorescein, rhodamine, coumarin, lanthanides

30. Protein labelling -One can design labelled systems: flexibility and versality. -The fluorophores can be attached to specific sites. -The protein is modified, which can alter its properties.

31. Summary -The luminescence phenomena -The definition of fluorescence and phosphorescence -Fluorescence parameters -How to measure fluorescence? -Applications (see also next week)

32. The linearity of the spectrophotometer; “stray light effect” Observation:

33. The principles of a monochromator

34. The linearity of the spectrophotometer; “stray light effect” The origin of the problem: not perfect monochromators! Optical grating And the second- third… harmonics!!!! 2 ; 3 …

35. substance I0I0 I 99% chosen and 1% second h. Absorbs only at the chosen ! 89% chosen and 1% second h. In the case of low absorption.

36. substance I0I0 I 99% chosen and 1% second h. Absorbs only at the chosen ! 1% chosen and 1% second h. In the case of high absorption.

37. Low absorption: I / I 0 = 90 / 100 = 0.9 real absorption = 89 / 99 ~ 0.9 The measured is close to the real one! High absorption: I / I 0 = 2 / 100 = 0.02 real absorption = 1 / 99 ~ 0.01 Relatively large deviation! The transmitted light: measured vs. real at the chosen

38. The resulting effect