FRET-FCS: Advancing comprehensive insights into complex biological systems

Förster resonance energy transfer (FRET) is a short-range distance-dependent photophysical phenomenon that allows the measurement of intra- and intermolecular distances through fluorescence detection. FRET measurements are sensitive to the movements of fluorescently labeled molecules as they produce fluorescence fluctuations. Fluorescence correlation spectroscopy (FCS) analyzes these fluctuations at faster and broader timescales (from picoseconds to seconds) compared with other techniques, unraveling the thermodynamic and kinetic properties of the system under study. Therefore, the combination of FRET and FCS (FRET-FCS) facilitates the analysis of molecular dynamics. Since its introduction, FRET-FCS has evolved into studying more sophisticated systems, requiring improvements in data acquisition and analysis. In this review, we discuss applications in the field of FRET-FCS that propose novel alternatives to overcome the inherent limitations of experimental setups. This work aims to promote using and enhancing FRET-FCS techniques to develop a comprehensive understanding of biological systems.