Abstract
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.
Highlights
With the development of the confocal microscope, fluorescence correlation spectroscopy (FCS)has been widely applied to analyze the diffusion coefficients of molecules and their conformational dynamics [1,2,3,4]
“Time-resolved FCS” [14], which is recognized as Fluorescence Lifetime Correlation Spectroscopy (FLCS) [15,16]
Yang and Xie built the conceptual basis of photon-by-photon analysis of excitation-detection delay time [20,21], which was later extended and experimentally demonstrated by
Summary
With the development of the confocal microscope, fluorescence correlation spectroscopy (FCS). FCS analyzes the correlation of single molecules through the temporal fluctuation of fluorescence signals that are detected from a tiny focal region of a microscope objective. In 2002, two groups independently published papers that were related to the utilization of fluorescence lifetime in FCS. Enderlein and his coworkers reported the concept and application of “Time-resolved FCS” [14], which is recognized as Fluorescence Lifetime Correlation Spectroscopy (FLCS) [15,16]. In FLCS, one uses fluorescence lifetime information to calculate the filter functions of all species in a heterogeneous system.
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