FluorescenceLifetimes培训课件.ppt

non-radiative processes: isolated molecules in “gas-phase” only internal conversion and intersystem crossing in condensed phase additional pathways due to interaction with molecular environment: excited state reactions, energy transfer,… ANS in water is ~100 picoseconds but can be 8 – 10 ns bound to proteins Ethidium bromide is 1.8 ns in water, 22 ns bound to DNA and 27ns bound to tRNA The lifetime of tryptophan in proteins ranges from ~0.1 ns up to ~8 ns Note: fluorescence lifetime tends to be shorter in more polar environment, because larger dipole moments of surrounding molecules can increase the efficiency of energy transfer The radiation lifetime tr = kf-1 is practically a constant for a given molecule The fluorescence lifetime t = k-1 = (kf + knr)-1 depends on the environment of the molecule through knr. Fluorescence quantum yield: is proportional to fluorescence lifetime. Addition of another radiationless pathway increases knr and, thus, decreases t and QY. However, the measurement of fluorescence lifetime is more robust than measurement of fluorescence intensity (from which the QY is determined), because it depends on the intensity of excitation nor on the concentration of the fluorophores. The fluorescence intensity I (t) = kf n*(t) is proportional to n*(t) and vice versa How to measure fluorescence lifetime ??? Time (or pulsed) domain Frequency (or harmonic) domain Molecules are excited by a very short pulse (close to a d-pulse) at t = 0 and the decay of florescence intensity is measured. Usually by Time Correlated Single Photon Counting (TCSPC) Excitation light is harmonically modulated with circular frequency w and so is the emission. Fluorescence lifetime can be deduced from the phase shift f and modulation m. t Time (or pulsed) domain Ideal single-exponential decay of fluorescence intensity (excited by a d-pulse at t = 0) The real fluorescence decay is a convolution with the profile of the excitation pulse The measured fluorescence decay is a c