Fluorescence lifetime imaging techniques: Frequency-domain FLIM

In general, the rate constant for the fluorescence pathway is invariant and time-independent. However, because the lifetime of an excited fluorophore is the inverse of the total rate of leaving the excited state, the measured lifetime will exhibit different values if the rate constant of a pathway other than fluorescence changes (e.g., a variable extent of dynamic quenching, or the presence of FRET). erefore, measured fluorescence lifetimes are excellent evaluators of the molecular environment of the fluorophore. As a result, FLIM has become popular for investigating the molecular environment in cells and tissues.