Fluorescence lifetime imaging microscopy (FLIM)
About this technique
Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence excitation lifetime of a fluorophore is measured at each position of a microscope image. Most biologically-active fluorophores typically have excitation lifetimes in the nanosecond timescale and this lifetime is independent of probe concentration and photobleaching.The factors that do affect fluorescence lifetimes include ion intensity, hydrophobic properties, oxygen concentration, molecular binding, and molecular interaction. Fluorescence resonance energy transfer (FRET), which is the energy transfer when two fluorescent proteins approach each other, affects the excitation lifetime. Therefore FLIM can be used to measure FRET. These variables mean fluorescence lifetime imaging is a particularly valuable technique to obtain information about the molecular environment of labeled macromolecules at each microscopically resolvable locationin living cells.