Photochromic fluorescence resonance energy transfer (pcFRET): formalism, implementation, and perspectives

Photochromic FRET (pcFRET), a member of the family of acceptor depletion FRET techniques (adFRET), embodies a general conceptual and experimental scheme based on a coupled system of a fluorescent donor and a photochromic acceptor. The procedure involves the reversible and cyclic spectroscopic depletion of the acceptor, and was initially conceived for the determination of FRET efficiency on a continuous, pixel-by-pixel basis in the microscopy of living cells. However, the modulation of donor fluorescence in pcFRET has implications for a wide range of applications. We present the formalism for quantitative interpretations of photostationary and kinetic data, from which the relevant kinetic rate constants and quantum yields for the cyclization and cycloreversion reactions of the photochromic acceptor can be derived. The scheme was applied to a model system consisting of a fluorescent donor (Lucifer Yellow) covalently bound to a diheteroarylethene acceptor. In a Perspectives section, we discuss photochromic probes, instrumentation issues, and the potential of pcFRET for analyzing chemical equilibria and kinetics, in the latter case with a new technique we have denoted Photochromic Relaxation Kinetics (pcRelKin).