A prototype instrument that uses fast switching photomultiplier tube (PMT) to acquire real-time time-resolved fluorescence lifetime data has been developed and tested at the University of Arizona. The PMT detector can collect fluorescence data at rates as high as 200 MHz without the production of switching transients. The circuit system also demonstrates a better signal integrity performance, a greatly improved bandwidth, and a faster transient response than any other approach to this problem. This system is capable of generating real-time fluorescence lifetime curves for molecules that have decay lifetimes as short as 80 ns.
Background:
Fluorescence lifetime imaging can be used to discern fluorophores that are otherwise indistinguishable with standard fluorescence imaging. In complex biological systems, where many kinds of fluorophores may be present, this type of imaging can become extremely useful. An unfortunate drawback to this method is that it takes a long time to acquire fluorescence lifetime data. Time limitations in detector response and signal acquisition necessitate the use of multiple excitation-decay cycles to build a histogram of lifetime data. Many dynamic biological systems change too rapidly for this type of imaging to be used, and for such cases, the concept of real-time time-resolved fluorescence lifetime imaging becomes very appealing. Having the ability to acquire lifetime data from a single excitation-decay cycle would make fluorescence lifetime imaging fast enough to monitor many short-lived biological processes. Fluorescence lifetime imaging would become a powerful imaging method not only for slowly changing biological samples, but also for high-speed dynamic systems.
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