Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications

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.

Microsecond Equilibrium Dynamics of Hairpin-Forming Oligonucleotides Quantified by Two-Color Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

“…Here, we address this issue by applying two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). ,, 2D FLCS enables us to determine the fluorescence lifetimes of coexisting species in a mixture sample and detect their change with a microsecond time resolution. Because the fluorescence lifetime of the donor fluorophore of a FRET pair is directly related to the FRET efficiency that is determined by the donor–acceptor distance, it is possible to clarify the microsecond conformational dynamics of biopolymers in equilibrium using 2D FLCS. , In the present work, we apply 2D FLCS to hairpin-forming RNA and DNA to compare their microsecond dynamics in a state-resolved manner under an equilibrium condition.…”

Scanning Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Conformational Dynamics of DNA Holliday Junction from Microsecond to Subsecond

“…For more quantitative analysis, we utilized the correlation matrix G(ΔT) that is related to the 2D lifetime correlation map with the following equation: 17,18,21,22 M T g Ta a ( ; , ) ( ) ( ) ( )…”

“…To obtain a comprehensive understanding of the dynamics of complex biomolecules, it is important to know both reaction kinetics and structural changes in a wide time window from submicrosecond to beyond seconds. − The single-molecule Förster resonance energy transfer (smFRET) method is one of the viable tools to elucidate molecular structure changes with extremely high sensitivity. − However, the application of conventional smFRET measurements has been limited to the time region down to several milliseconds because of the difficulty in collecting a sufficient number of photons to evaluate the FRET efficiency in a short bin time, although several special techniques that achieve sub-100 μs time resolution have been reported. − Two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) developed by our group successfully solved this problem of the time resolution in smFRET. − In 2D FLCS, the fluorescence photons from the FRET donor dye are detected by time-resolved measurements. Then, the temporal correlation of the fluorescence lifetime is analyzed using a 2D map constructed for a certain delay time, in which structural dynamics gives rise to cross-peaks reflecting interconversion between different conformations with distinct FRET efficiencies.…”

“…We employ a recently developed extension of 2D FLCS that incorporates the analysis of the acceptor fluorescence, i.e., two-color 2D FLCS. , In two-color 2D FLCS, the states exhibiting different FRET efficiencies are determined as independent fluorescence components that are required to reproduce the 2D correlation maps that are constructed from the donor and acceptor fluorescence photons recorded with time-tagged TCSPC. Detailed procedures and interpretations of two-color 2D FLCS are described in our previous paper − as well as in the Supporting Information.…”

Single Molecule Fluorescence Spectroscopy and Imaging: Advanced Methods and Applications in Life Sciences

“…The applications of FLCS include quantification of protein-protein interactions [165], reduction of spectral crosstalk in live-cell studies [166], investigation of lifetime changes of a fluorophore in close proximity to silver nanoparticles [167], FLCS experiments in conjunction with lifetime tuning [168] for studying diffusion in SLBs, investigation of DNA compaction by spermine [169], proton transfer reactions [105], and, along with FRET, quantification of kinetic rates of interconversion in Syntaxin 1 [170]. Furthermore, we furnish the details of an interesting extension of FLCS, known as two-dimensional FLCS [171], and discuss also the utilization of FLCS in STED microscopy [106].…”