FLIM is a microscopy technique which can be used as a method for quantification of molecular interactions and a very elegant readout to measure FRET (Fluorescence Resonance Energy Transfer) i. e. the proximity of two fluorophores within a cell. The fluorescence lifetime parameter readout is independent on fluorophore concentration – as opposed to fluorescence intensity - and thus does not need calibration measurements. Calibration measurements are tedious in cells and impossible in live cell samples. Therefore, FLIM is a very robust way to measure protein-protein interactions. It is the only way to measure such interactions in live cells and it is essential for studying e. g. kinase signaling in live cells because there is no live cell compatible fluorescent probe available for protein phosphorylation.
Fluorescence intensity image (left panel). Fluorescence lifetime image (right panel)
The principle of FLIM
Fluorescence lifetime t is sensitive to radiative (kF) as well as non-radiative processes (quenching, energy transfer….), but independent of concentration, scattering, absorbance.