ABSTRACT
This chapter explains the different types of fluorescence lifetime imaging microscopy (FLIM) instrumentation with an emphasis on analysis techniques, applications, and their challenges. It introduces the basic concepts of fluorescence lifetime and advance to the different instrumentation schemes used to realize FLIM in the biological research laboratory, common methods to analyze FLIM data, and representative FLIM applications. FLIM imaging with superresolution techniques such as stimulated emission depletion microscopy enables exploiting Förster resonance energy transfer to study protein interaction localized well below the traditional limit. Another noticeable problem in FLIM is the lack of effective standards to validate and estimate the current FLIM techniques. Fluorescence lifetime is the average time spent by an excited molecule in its excited state before returning to the ground state by the emission of a fluorescent photon.
