FLIM- Fluorescence Lifetime Imaging Microscopy

FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique inconfocal microscopy, two-photon excitation microscopy, and multiphoton tomography. The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample.

Since the fluorescence lifetime of a fluorophore depends on both radiative (i.e. fluorescence) and non-radiative (i.e. quenching, FRET) processes, energy transfer from the donor molecule to the acceptor molecule will decrease the lifetime of the donor. Thus, FRET measurements using FLIM can provide a method to discriminate between the states/environments of the fluorophore. In contrast to intensity-based FRET measurements, the FLIM-based FRET measurements are also insensitive to the concentration of fluorophores and can thus filter out artifacts introduced by variations in the concentration and emission intensity across the sample.