FLIM measurements are carried out with Leica TCS SP8 SMD with FLIM configuration.
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. The emission of a fluorescence photon from a fluorophore does not occur at a fixed time. Instead, a distribution of times is observed, which can be described by an exponential decay function. The characteristic time constant of this decay, the fluorescence lifetime, is in the range of a few picoseconds (10-12 s) to several tens of nanoseconds (10-9 s).
This lifetime is a characteristic parameter of each fluorescent dye that may change with its micro-surrounding or its conformational state. Lifetime information probes the molecular environment for its composition, such as ion concentration, pH, lipophilicity or the binding to other molecules.
FLIM combines lifetime measurements with imaging: lifetimes obtained at the pixel-level are color-coded to produce images. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its nano-environment. This way an additional dimension of information is obtained.