7. Chemical Structure of the Chromophore

8. Biosynthesis of the Chromophore Critcial dehyrogenation reaction to juxtapose aromatic group with imidazlinone

9. Excitation and Fluorescence Emission Spectra of GFP

10. Förster cycle Hydroxyl form Phenol-OH Phenolate Phenol-O - Protonated excxited Excited phenolate

12. wt egfp S65t t203i Yfp S65g, t203y Cfp F64l, s65t, y66w Bfp Y66h, y145f

13. The Beta-Can Structure

14. Topology of Folding

15. Cysteins

16. The Environment of the Fluorophor

24. Light microscopy - fancy techniques. FLIM - fluorescence life-time imaging; used to study interactions between molecules in living cells. Used in conjunction with FRET. FLIM measures the fluorescent life-time of the FRET donor. Decay of the activated FRET donor depends on its environment and association with an acceptor. FLIM data can be used to calculate true FRET efficiencies and confirm molecular interaction in living cells. This requires a pulse laser and so is routinely performed on a multiphoton lm. FCS - fluorescence correlation spectroscopy; monitors random motion of fluorescently labelled molecules in a defined cell volume, which is irradiated by a focussed laser beam. Monitored changes give information about rates of diffusion and so apparent mass. If mass is known - likely association with cell compartments can be implied. Hence this is versatile for dynamics studies in live cells. Uses FCS module attached to routine confocal microscope