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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.

european biophysics journal

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations