Time-resolved fluorescence spectroscopy detectors

E. Gratton, B. Feddersen, M. vandeVen, Parallel acquisition of fluorescence decay using array detectors, in Time-Resolved Laser Spectroscopy in Biochemistry II (J. R. Lakowicz, ed.), Proc. SPIE 1204, 21-25(1990). 

As a result of enormous development in the technology and production of pulse lasers, laser diodes, detector systems, and powerful computers in recent decades, steady-state and time-resolved fluorometers now belong to the standard equipment of biochemical and macromolecular laboratories. For example, there are apparatuses combined with microscopes that are suitable for time-resolved fluorescence measurements of individual organelles in living cells. However, the widespread use of fluorescence techniques generates certain danger, which is connected with their routine use. We would like to point out that the fluorescence spectroscopy is an indirect technique and that the interpretation of results needs great care and precaution. It almost always requires additional information on the system. 

Because of the underlying photophysics, fluorescence lifetimes are intrinsically short, usually on the order of a few nanoseconds. Detection systems with a high timing resolution are thus required to resolve and quantify the fluorescence decays. Developments in electronics and detector technology have resulted in sophisticated and easy to use equipment with a high time resolution. Fluorescence lifetime spectroscopy has become a popular tool in the past decades, and reliable commercial instrumentation is readily available. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

Spectroscopy of single molecules is based on fluorescence correlation, photoncounting histograms, or burst-integrated-lifetime techniques. Each case requires recording not only the times of the photons in the laser period, but also their absolute time. Modem time-resolved single molecule techniques therefore use almost exclusively the FIFO (time-tag) mode of TCSPC. The FIFO mode records all information about each individual photon, i.e. the time in the laser pulse sequence (micro time), the time from the start of the experiment (macro time), and the number of the detector that detected the photon (see Sect. 3.6, page 43). 

---