Instrumentation flashlamps

Given the low sensitivities of photodiodes, one may question their use as the start detector in the TCSPC instrument schematically represented in Figure 4.7. In this case the light source was a laser, which could be readily focused onto the small active area of a photodiode. Photodiodes are not used as the reference detector with flash-lamps because of their low sensitivity. When the light source is a flashlamp, the detector is either a PMT or a wire which acts as an antenna to detect the RF leakage during the lamp pulse. 

In the previous chapter we presented an overview of protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties depend on protein structure. We now extend this discussion to include time-resolved measurements of intrinsic protein flu( scence. Prior to 1983, most measurements of time-resolved fluorescence were performed using TCSPC. The instruments employed for these measurements typically used a flashlamp etcitation source and a standard dynode-chain-type PMT. Such instruments provided instrument response functions with a half-width near 2 ns, which is comparable to thedecay time of most proteins. The limited repetition rate of the flashlamps, near 20 kHz. resulted in data of modest statistical accuracy, unless the acquisition times were excessively long. Given the complexity of protein intensity and anisotropy decays, and the inherent difficulty of resolving multiexponential processes. ii was difficult to obUun definitive information on the decay kinetics of proteins. 

Diuing the past decade, the instrumentation for time-resolved fluorescence of proteins has advanced dramatically. The flashlamp light sources have been replaced by hi -repetition-rate (MHz) picosecond dye lasers, which provide both higher excitation intensities and raon rapid data acquisition. The dynode-chain PMTs have been replaced by MCP detectors, which provide much shorter single-photoelectron pulse widths than a dynode chain PMT. In con nnation. the new light sources and detectors provide instniment r ponse functions with half-widths near 100 ps, so that picosecond resolution can now be obtained. 

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