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Förster Resonance Energy Transfer , abbreviated  FRET , is a fluorescence phenomenon, which can be used to study  and map co-localizations and dynamics of co-localizations at nanometer precision on a light microscope. FRET has been  described as a  spectroscopic ruler . The efficiency of the radiationless energy transfer from an excited chromophore, the   donor , to another chromophore, the  acceptor , the excitation energy of which approximately matches the energy to be  released by the donor, is dependent on the sixth power of the mutual distance between the two molecules in space.  We propose a new, non-destructive technique for measuring FRET quantitatively and at high spatial and temporal  resolution on a laser scanning microscope:  Two laser beams of wavelengths suitable for the mutually exclusive excitation of the donor and the acceptor, the  donor  beam  and the  acceptor beam , respectively, are intensity modulated by means of two electro optical modulators  (EOM). The modulation patterns are rectangular at duty cycle ½. The modulation frequencies differ slightly. The  acceptor beam is saturating the acceptor so that it cannot accept energy from the donor. The saturation is modulated in  the same way as the acceptor beam. Since the donor beam also is modulated, though at a frequency slightly different  from that of the acceptor beam, the intensity of the released donor fluorescence is modulated with the beat frequency of  the frequencies of the two laser beam modulations and can be detected and interpreted in quantitative terms by means of  a lock in amplifier.Copyright 2011 Society of Photo-Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited

Proposal of a new method to measure FRET quantitatively in living or fixed biomedical specimens on a laser microscope