FRET, Fluorescence Resonance Energy Transfer
FRET is the abbreviation for "Förster or Fluorescence Resonance Energy Transfer" and is a useful tool to quantify molecular dynamics like interactions of two fluorophores by microscopy. The proteins under investigation are labelled with donor fluorophores or acceptor fluorophores. Interaction between the two fluorophores is accompanied by direct energy transfer from donor to acceptor (FRET). When FRET occurs, it means that the two proteins of interest are in such close proximity that they can interact with each other.
During FRET, a quantum of energy is transferred from a donor fluorophore to an acceptor fluorophore in a nonradiative process. So, in case of no FRET, the donor fluorophore is excited and emits photons. The acceptor fluorophore does not emit photons, because it is not excited. In case of FRET, the donor fluorophore is excited, but in stead of emitting all its energy as photons, it transfers some of its energy to the acceptor fluorophore that becomes excited and emits light.
More information can be found at the Wikipedia site: FRET.
Summarising, in case of no FRET only the donor fluorophore emits photons, and in case of FRET both donor and acceptor emit photons.
technologies
Measurement methods FRET
FRET can be measured in several ways, like the ratiometric method FRET and the FLIM-FRET method. Both methods measure the FRET efficiency, i.e. the efficiency in which the donor fluorophores transfer their energy non-radiatively to the acceptor fluorophores that can be excited by this transferred energy.
FRET pairs
The emission spectrum of the donor fluorophore has to overlap the excitation spectrum (absorbance) of the acceptor fluorophore. Some examples are BFP-YFP, CFP-YFP, GFP-DsRed, GFP-Cy3, GFP-mOrange, YFP-RFP, and Cy3-Cy5.
FRET efficiency
The FRET efficiency indicates the percentage of the excitation photons that contribute to FRET. It depends strongly (sixth-power relationship) on the distance between the donor and acceptor fluorophores. The lifetime is inversely proportional to the FRET efficiency, thus the higher the efficiency the lower the lifetime of the donor fluorophore becomes.
However, the FRET efficiency in one pixel of your image does not give exact conclusions about the interactions between fluorophores. Namely, in case of 50% FRET efficiency in one pixel of your image, it is possible that 50% of the donor fluorophores have had 100% energy transfer to acceptor fluorophores, but it is also possible that 100% of the donor fluorophores have had 50% energy transfer to acceptor fluorophores.
FRET only occurs if…
1) the donor fluorescence emission spectrum overlaps with the acceptor absorbance.
2) the donor and acceptor fluorophores are in close proximity (i.e. 1 - 9nm, which is at the scale of protein size).
3) the transition dipole moments of the donor and acceptor fluorophores are not perpendicular.