Förster Resonance Energy Transfer (FRET) can occur when two fluorophores are very close together (e.g. within ~10nm) and have complementary spectral profiles and orientations. Generally speaking the “donor” fluorophore is excited and then transfers energy directly to the “acceptor” which then emits a photon. Thus the emission spectrum of the donor should overlap significantly with the excitation spectrum of the acceptor. There are several ways to perform FRET studies, but one of the most rigorous and flexible techniques for quantifying FRET is through fluorescence lifetime imaging (FLIM). FLIM involves measuring the lifetime of a fluorophore using a pulsed laser, and the lifetime will decrease when FRET occurs. Thus, FLIM involves permits direct quantification of FRET and is not susceptible to artefactual measurements possible with some other FRET techniques.