Abstract
In this review, we give a historical view of how our research in the development and use of fluorescence correlation spectroscopy (FCS) and related techniques has its roots and how it originally evolved from the pioneering work of Manfred Eigen, his colleagues, and coworkers. Work on temperature-jump (T-jump) experiments, conducted almost 50 years ago, led on to the development of the FCS technique. The pioneering work in the 1970s, introducing and demonstrating the concept for FCS, in turn formed the basis for the breakthrough use of FCS more than 15 years later. FCS can be used for monitoring reaction kinetics, based on fluctuations at thermodynamic equilibrium, rather than on relaxation measurements following perturbations. In this review, we more specifically discuss FCS measurements on photodynamic, electronic state transitions in fluorophore molecules, and on proton exchange dynamics in solution and on biomembranes. In the latter case, FCS measurements have proven capable of casting new light on the mechanisms of proton exchange at biological membranes, of central importance to bioenergetics and signal transduction. Finally, we describe the transient-state (TRAST) spectroscopy/imaging technique, sharing features with both relaxation (T-jump) and equilibrium fluctuation (FCS) techniques. TRAST is broadly applicable for cellular and molecular studies, and we briefly outline how TRAST can provide unique information from fluorophore blinking kinetics, reflecting e.g., cellular metabolism, rare molecular encounters, and molecular stoichiometries.
Highlights
In 1967, Manfred Eigen, together with Ronald G.W
The first attempts to use fluorescence as the readout in fluctuation experiments illustrated that such analyses of systems under thermodynamic equilibrium can offer an attractive alternative to relaxation measurements, studying the response in a sample following some external perturbation
Correlation analyses can be performed on fluorescence intensities recorded in e.g., different spectral channels [fluorescence cross-correlation spectroscopy, FCCS (Ricka and Binkert 1989; Schwille et al 1997)], spatial locations [image correlation spectroscopy, ICS (Brinkmeier et al 1999; Petersen et al 1993; Digman et al 2005)], or based on fluorescence lifetime changes [fluorescence lifetime correlation spectroscopy, FLCS (Benda et al 2006)]. It is beyond the scope of this review to discuss the full range of molecular dynamic processes that can be studied by Fluorescence correlation spectroscopy (FCS)
Summary
FCS is not limited to analyses of the number and diffusion properties of fluorescent molecules. Correlation analyses can be performed on fluorescence intensities recorded in e.g., different spectral channels [fluorescence cross-correlation spectroscopy, FCCS (Ricka and Binkert 1989; Schwille et al 1997)], spatial locations [image correlation spectroscopy, ICS (Brinkmeier et al 1999; Petersen et al 1993; Digman et al 2005)], or based on fluorescence lifetime changes [fluorescence lifetime correlation spectroscopy, FLCS (Benda et al 2006)] It is beyond the scope of this review to discuss the full range of molecular dynamic processes that can be studied by FCS. In a more general case, the separation of G(τ) into two factors as in Eq 3 is possible for reactions in which the diffusion of the reactants and product molecules is much slower than the chemical relaxation time(s) and/or the diffusion coefficients of all fluorescent species can be considered equal (Palmer and Thompson 1987). How monitoring of ion-sensitive fluorophores by FCS offers an alternative way of monitoring proton exchange kinetics and how this approach can be used to investigate protonation kinetics at biological membranes
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