FRET and FCS--friends or foes?

Abstract

Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) are both scientific concepts that are frequently discussed in the context of single-molecule fluorescence techniques. In contrast to FCS, FRET is strictly not a technique but a photophysical phenomenon, which can be employed in combination with any method that probes fluorescence intensity or lifetime. Thus, the combination of FCS with FRET is possible and—although these concepts are quite often treated as alternative approaches, particularly for the analysis of biological systems—also quite attractive. However, under certain circumstances, for example, for applications of fluorescence cross-correlation spectroscopy, FRET effects can cause significant complications for quantitative data analysis, and careful calibration has to be carried out to avoid FRET-induced artifacts. This can be most elegantly done if alternating excitation schemes such as PIE (pulsed interleaved excitation) are employed. In this minireview, we discuss the potential and the caveats of FCS combined with FRET and give a short record on successful and promising applications.