Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

Abstract

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector after pulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy

“…Additionally, raw autocorrelation curves were filtered by Fluorescence Lifetime Correlation Spectroscopy (FLCS) function to remove the background noise and after-pulsing artifacts. 82 …”

Lifetime-Weighted FCS and 2D FLCS: Advanced Application of Time-Tagged TCSPC

“…Such reinterpretation provides a coherent overview of the existing methods, which can lead us to further development of a new analysis. For example, FLCS developed by Enderlein and coworkers [5][6][7][8] can be understood in the framework of M(ΔT ) as follows. In FLCS, it is assumed that the complete prior knowledge of the fluorescence decay components is available.…”

“…Therefore, they allow us to study fluorescence lifetime fluctuations with a time resolution down to tens of nanoseconds. Fluorescence lifetime correlation spectroscopy (FLCS) [5][6][7][8] is a useful technique when one has information about the fluorescence decay of each species in advance. In this case, FLCS utilizes the microtime information of each detected photon to infer the source of the photon, i.e., the species from which the photon is emitted.…”

Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis

“…The timing instability of SPADs is problematic, not only for FLIM, but also for other techniques utilizing fluorescence lifetime. 9,10 The simple calibration method demonstrated in this study allows us to improve the accuracy of the fluorescence lifetime determined with SPADs and enables us to fully take advantage of this type of the detector.…”

“…These temporal data are very informative in single-molecule spectroscopy and fluorescence correlation spectroscopy, and their usefulness has been demonstrated in several applications. [5][6][7][8][9][10] In the present study, this time-tag mode was utilized for examining the photon interval dependence of the temporal response of SPADs.…”