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Record and analyze fluorescence lifetime images.
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Intensity-based FRET

Forster Resonance Energy Transfer (FRET) efficiency E indicates the percentage of the excitation photons that contribute to FRET and is defined as:

where τDA is the fluorescence lifetime of the donor in the presence of an acceptor, and τD in the abscence of an acceptor. As you can see, the more FRET occurs, the more decrease in donor fluorescence lifetime.

FRET strongly depends on the distance between the donor and acceptor fluorophores (sixth-power relationship). Fluorescence lifetime of a fluorescent molecule is inversely proportional to its FRET efficiency, thus the higher the FRET efficiency the lower the fluorescence lifetime of the donor molecule will be.

The efficiency also depends on the donor-to-acceptor separation distance R with an inverse 6th order law due to the dipole-dipole coupling mechanism:

with R being the distance between donor and acceptor pair and R0 being the Förster distance between donor and acceptor at which the FRET efficiency is 50%.

FRET efficiency in a single pixel of an image, does not give exact conclusions about the interactions between fluorophores. The entire 2D image gives a better overview of the interactions that occur. For example: in case of 50% FRET efficiency in a single pixel, it could be possible that 50% of the donor fluorophores have had 100% energy transfer to acceptor fluorophores, but it also could be possible that 100% of the donor fluorophores have had 50% energy transfer to acceptor fluorophores.