Photobleaching FRET Microscopy

This chapter discusses the use of photobleaching in Forster resonance energy transfer (FRET) microscopy. Photobleaching is a process in which a fluorophore is irreversibly destroyed following repeated excitation. Jovin, Arndt-Jovin, and colleagues recognized that photobleaching could be used as a tool to measure Forster resonance energy transfer (FRET). They introduced two experimental methods that exploit photobleaching as a method to follow changes in the donor resulting from FRET. First, donor photobleaching FRET (donor pbFRET) measures the kinetics of the photobleaching of donor fluorescence in the presence and the absence of the acceptor. Second, acceptor photobleaching FRET (acceptor pbFRET) allows the direct measurement of donor dequenching on a single sample by eliminating the acceptor. Both these methods can be used to directly calculate energy transfer efficiencies, without the need for extensive corrections required to quantify sensitized emission measurements of FRET. They also do not require specialized instrumentation, making them easily available to investigators with access to research-grade digital fluorescence microscopes. Acceptor pbFRET is widely used as a method of choice for quantitative FRET measurements, especially with confocal microscopy. The chapter also discusses the application of pbFRET to the study of membrane microdomains.