Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications

We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the microenvironment and can be a “molecular ruler” when combined with FRET. Utilization of fluorescence lifetime in 2D FLCS thus enables us to quantify the inhomogeneity of the system and the interconversion dynamics among different species with a higher time resolution than other single-molecule techniques. Recent applications of 2D FLCS to various biological systems demonstrate that 2D FLCS is a unique and promising tool to quantitatively analyze the microsecond conformational dynamics of macromolecules at the single-molecule level.