In the intensity-based Forster Resonance Energy Transfer (FRET) method, change in emission intensities from donor and acceptor fluorophores is measured. During FRET, the amount of emitted photons (emission intensity) from the donor fluorophore decreases and the emission intensity from the acceptor fluorophore increases. The FRET efficiency is basically calculated from the ratio of emission intensities from donor and acceptor before and after FRET occurrence.
To obtain accurate FRET data by sensitized emission, three images have to be acquired:
- Donor excitation with donor emission,
- Donor excitation with acceptor emission,
- Acceptor excitation with acceptor emission.
The major advantage of this method over fluorescence lifetime imaging microscopy (FLIM)—which is a donor-based FRET detection—is that it can be carried out with standard wide-field or confocal fluorescence microscopes that are available in most laboratories. Moreover, it yields additional data on the acceptor population. However, quantitative sensitized emission requires significant attention for corrections and calibration, whereas FLIM-based FRET techniques are inherently quantitative from first physical principles. [Ref. Gadella TW Jr., FRET and FLIM techniques, 33, 2008]