Lecture 3 Fluorescence microsopy I CCAM讲座3荧光显微镜我CCAM.pptVIP

Anisotropic sample - Fluorescent intensity is dependent on the polarization _and_ the orientation of the molecules Isotropic sample Fluorescent intensity is independent of excitation polarization Fluorescence is polarized if the excitation is polarized. Fluorescence anisotropy r 0.4 Microscopic Measurements of Anisotropy Use Small Numerical Aperture r = r0 / ( 1 + ? / ? ) Fluorescence Resonance Energy Transfer (FRET) F?rster Radius The distance at which energy transfer is 50% efficient (i.e. 50% of excited donors are deactivated by FRET) is defined by the F?rster Radius (R0). Applications of FRET in Biology Survey of FRET-Based Assays Protease activity Calcium Ion measurements cAMP Protein tyrosine kinase activity Phospholipase C activity Protein kinase C activity Membrane potential FRET probes conformational changes Different conformation gives Different FRET signature FRET increases In both cases Inter and Intramolecular Forms of FRET with Proteins CFP-YFP good combo Protein-Protein Interactions In cytoplasm and membranes No FRET for No overlap of donor emission, acceptor absorption When FRET Occurs No FRET for Orthogonal dipole orientation No FRET for molecules more than 10 nm apart Number of FRET Publications since 1989 Fluorescence Resonance Energy Transfer - Detection of Probe Proximity R0 typically 40-50 Angstroms 50% transfer Typical Values of Ro Donor Acceptor Ro (?) Fluorescein Tetramethylrhodamine 55 IAEDANS Fluorescein 46 EDANS DABCYL 33 Fluorescein Fluorescein 44 BODIPY FL BODIPY FL 57 Fluorescein QSY 7 dye 61 Cy3 Cy5 53 CFP YFP 50 green red GFPs and other colored “FPs have transformed FRET microscopy FRET Considerations: 1. Spectral overlap 2. Chromophore orientations 3. Distance dependence (Eff. ? 1/R6) 4. How to quantify? Practical Challenges to FRET Quantitation Emission from A contaminates D channel (filters) Emission from D contaminates A channel Unknown labeling levels for D and A Signal variation due to bl