The fluorescence lifetime is a measure of how long a fluorophore remains on average in its excited state before returning to the ground state by emitting a fluorescence photon. It depends on the molecular composition and nano-environment of the fluorophore.

FLIM combines lifetime measurements with imaging: lifetimes obtained for each image pixel are color-coded to produce additional image contrast. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its biochemical status or nano-environment. A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well.

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FLIM-FRET can be measured on the Leica SP8-SMD and analyzed with SymPhoTime 64.