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

Abstract

“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.