23] Fluorescence resonance energy transfer imaging microscopy

Abstract

This chapter describes the fluorescence resonance energy transfer (FRET) imaging microscopy. RET is a process by which a donor fluorophore (D) in its excited state may transfer its excitation energy to a nearby chromophore (acceptor, A). The process is nonradiative (not mediated by a photon) and is achieved through dipole–dipole interactions. In principle, for such a transfer to occur, several major criteria must be satisfied. First, the donor molecule must have an emission spectrum that overlaps the absorption spectrum of the acceptor molecule. The second criterion that needs to be satisfied is that the donor and acceptor molecules must be within 10–100 Å of each other. The efficiency of energy transfer between donor and acceptor molecules falls off as the sixth power of the distance separating the donor and acceptor molecules. Another criterion that must be met is that the fluorescence lifetime of the donor molecule must be of sufficient duration to allow FRET to occur. Continued research in this area is needed to determine optimal approaches for the measurement of FRET and the development of more refined algorithms for the analysis of data.