Hi Weidong,

Förster Resonance Energy Transfer (FRET) is an excited state process that occurs between a fluorophore and another molecule. FRET can be said to occur when the emission spectrum of the fluorophore, termed the donor, overlaps with the absorption spectrum of another molecule, known as the acceptor.

In FRET, the donor molecule, i.e. the fluorophore, does not release a fluorescent photon as there is no radiative transfer of energy between the donor and acceptor. Instead, the incident energy absorbed by the donor is transferred through a non-radiative mechanism by way of a virtual photon.

FRET has incredibly sensitive distance-dependence and as such is termed the 'spectroscopic ruler'.

The image I've included I sourced from:

http://www.calctool.org/CALC/chem/photochemistry/fret.png

FRET causes there to be an emission peak at a longer wavelength than one would expect. As such, if you have clearly defined the excitation and emission spectra for your known fluorophores then one can expect FRET to be occurring if there is a third, longer emission wavelength.

Derek.

I'd just like to add that FRET occurs when there is a distance separation of 10 nm at most between dyes. If you use diffraction limited light microscopy, confocal microscopy, or even supperresolution, up to date the size of the illuminated PSF is much larger than the distance ranges for FRET, and even though you catch that colocalized voxel for 300 frames, the question is, are they interacting (distance less than 10 nm) or are they just occupying the same voxel for 600 frames.

In light of that, what is the frame rate? Is it a live or fixed cell measurement? etc etc

This is exactly why (Fluorescence Lifetime IMaging (FLIM) microscopy is used to quantify FRET. changes in intensities of both dyes are hard to interpret, but one thing for sure, if indeed this voxel includes FRET, the donor dye lifetime will decrease and that of the acceptor dye will increase..