Single-Molecule FRET

This chapter focuses on the practical aspects of single-molecule Förster resonance energy transfer (FRET) experiments. Ideal dyes for single-molecule fluorescence studies should possess several characteristics including photostable; bright (high extinction coefficient and quantum yield of emission); stable, showing little intensity fluctuation; excitable and emit in the visible spectrum; relatively small so that they introduce minimum perturbations to the host molecule; and commercially available in a form that can be conjugated to biomolecules. Ideal single-molecule FRET dyes have large Stokes' shifts to minimize the amount of donor emission that bleeds through into the acceptor channel and to reduce the amount of direct excitation of the acceptor by the laser. There are two classes of fluorescence microscopy tools for single-molecule fluorescence studies. The first involves point detection using a single-element detector that is most commonly in the confocal microscope configuration, whereas the second class of tools for single-molecule studies consists of wide-field microscopy with a two-dimensional detector such as a charge-coupled device (CCD) camera.