Fluorescence lifetime imaging microscopy (FLIM) is a fluorescence-based imaging technique that not only measures intensity differences on a pixel basis, but also detects the decay dynamics of excited fluorophores.
This allows differentiation between fluorophores that overlap in their excitation and emission spectra, but exhibit differences in fluorescence decay dynamics. Additionally, some fluorophores show specific changes in fluorescence lifetimes dependent on their microenvironment. Thus, FLIM is ideally suited to measure changes in chloride concentration, local pH, and other factors. Moreover, Förster Resonance Energy Transfer (FRET) sensors change their lifetime dependent on activity, which allows additional analyses
