Lambert Instruments

Förster / Fluorescence Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from a molecule in the excited state (donor) to a molecule in the ground state (acceptor). A fluorescent donor molecule can return to the ground state by losing its energy through emission of a photon (fluorescence), or by transferring its energy to a nearby (1 - 9nm) acceptor molecule (FRET). Compared to a molecule that exhibits no FRET, the donor has more options to lose its energy. Therefore, it returns faster to the ground state, which decreases its lifetime. 

FRET efficiency
The FRET efficiency E is defined as E = 1 - ( DA / D ) where DA and D are the donor fluorescence lifetimes in the presence and in absence of an acceptor fluorophore respectively. So, the more FRET, the more decrease in donor lifetime.

Advantages FLIM-FRET
FLIM-FRET has a lot of advantages above FRET detection without FLIM. One of these advantages is that FRET measurements done by FLIM are more robust and quantitative than the FRET measurements done by e.g. sensitised emission FRET. The fluorescence lifetime is independent on the intensity of the fluorophores, and therefore one can use any cell in the sample for FLIM-FRET experiments. Another advantage is that only the lifetime of the donor fluorophore has to be measured. So, the acceptor fluorophore might have inefficient emission (even might be a quencher). Moreover, because the acceptor fluorophore does not need to be measured, the measurements can be done a lot faster and easier. Furthermore, no corrections are needed for donor fluorophore emission bleed through in the acceptor emission channel.

Acceptor Photobleaching
By photobleaching the acceptor fluorophore during FRET, the non-radiative transfer of energy from the donor to the acceptor decreases. Therefore, the donor fluorophore loses less energy and the fluorescence lifetime of the donor fluorophore increases as compared to FRET without photobleaching the acceptor. Only when the acceptor has been bleached completely, the lifetime of the donor fluorophore will be similar to the situation of no FRET occurrence (donor only). However, be aware of some pitfalls.

Enhanced Acceptor Fluorescence (EAF)
In case the donor and acceptor fluorophores are both excited with the same excitation light wavelength, e.g. in case of the FRET pair GFP-YFP, a special kind of FRET can be detected. Namely, the average lifetime that is calculated is the contribution of both donor and acceptor fluorophores. Taking GFP and YFP as example, GFP has a small lifetime compared to YFP. When no FRET is occurring, the average lifetime is measured of both GFP and YFP that are both excited by the 480nm wavelength light source. However, when FRET occurs, the energy of the GFP proteins transfers non-radiatively to the YFP proteins, so relatively more YFP emission (with a long lifetime) is taken into account. So, the average lifetime increases in stead of decreases, as is normally the case when you measure only the lifetime of the donor fluorophore.