Fluorescence Specroscopy 朝陽科技大學荧光光谱朝陽科技大學.ppt

Fluorescence Spectroscopy Part I. Background Characteristics of Excited States Energy Lifetime Quantum Yield Polarization Stokes shift The Stokes shift is the gap between the maximum of the first absorption band and the maximum of the fluorescence spectrum Polarization Molecule of interest is randomly oriented in a rigid matrix (organic solvent at low temperature or room temperature polymer). And plane polarized light is used as the excitation source. Degree of polarization is defined as P Inner Filter Effect At low concentration the emission of light is uniform from the front to the back of sample cuvette. At high concentration more light is emitted from the front than theback. Since emitted light only from the middle of the cuvette is detected the concentration must be low to assure accurate FF measurements. The structure of GFP : eleven-strand beta-barrel wrapped around a central alpha-helix core. This central core contains the chromophore which is spontaneously formed from a chemical reaction involving residues Ser 65, Tyr 66, and Gly 67 (SYG) There is cyclization of the polypeptide backbone between Ser 65 and Gly 67 to form a 5-membered ring, followed by oxidation of Tyr 66. The high quantum yield of GFP fluorescence probably arises from the nearly complete protection of the fluorophore from quenching water or oxygen molecules by burial within the beta-barrel. Resolution of the contributions of individual tryptophan residues in multi-tryptophan proteins. I(?,t)=??i(?)exp(-t/?i) i ?1=2ns, ?2= 5ns ?1=2ns ?2=5ns t (ns) Fluorescence intensity (A.U.) Fluorescence intensity (A.U.) wavelength (nm) ? ?em Example Time-resolved protein fluorescence Isolated from the Pacific jellyfish Aequorea victoria and now plays central roles in biochemistry and cell biology due to its widespread use as an in vivo reporter of gene expression, cell lineage, protein - protein interactions and protein trafficking One of the most important attributes of GFP which makes it so useful i