FRET is a well-established fluorescence-based technique to detect and quantify biomolecular interactions, including readouts of genetically expressed FRET biosensors. FRET measurements require the biomolecules of interest to be labelled with complementary fluorophores, such that the emission spectrum of one fluorophore (called the “donor”) overlaps with the excitation spectra of the second fluorophore (the “acceptor”). When these complementary fluorophores are in close proximity (<~10 nm) there can be an efficient energy transfer process resulting in a reduction in emission from the donor fluorophore and fluorescence emission of the acceptor that is only excited via the energy transfer. The efficiency of this energy transfer scales inversely with the 6th power of the fluorophore separation and is effectively negligible for donor-acceptor distances >~10 nm. Thus detection of FRET is a strong indication of interaction of the labelled biomolecules. Using genetically expressed fluorophores (such as CFP with YFP or GFP with mCherryFP) to label specific proteins, FRET is widely used to map protein-protein interactions in live cells and to readout FRET biosensors, which are molecular constructs that are labelled with both donor and acceptor fluorophores.

FRET can be detected through a wide range of changes in fluorescence parameters of donor of acceptor fluorophores but the two most common FRET readouts are spectral ratiometric FRET and fluorescence lifetime imaging (FLIM). Spectral ratiometric FRET measures the fluorescence intensity of the donor and acceptor emission. The resulting FRET ratio generally provides a qualitative readout of FRET and requires careful calibration to account for spectral cross-talk and other potential artefacts, particularly when the relative donor and acceptor fluorophore concentrations are unknown. Spectral ratiometric FRET is most robust when applied to single molecule FRET biosensors. With FLIM, only the donor emission is measured and FRET is indicated by a decrease in the donor fluorescence lifetime since the energy transfer provides an additional relaxation pathway for the excited donor fluorophores. Fluorescence lifetime-based measurements are therefore independent of donor/acceptor stoichiometry and also of spectral properties of the sample or instrumentation. Furthermore, by fitting the donor fluorescence decay profiles to a double exponential decay model, it is possible to determine the fraction of the donor fluorophore population that is undergoing FRET (and therefore the fraction of labelled biomolecules that are interacting).