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

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.

Three-State DNA Hairpin Conformational Dynamics Revealed by Higher-Order Fluorescence Correlation Spectroscopy

“…A brief discussion of second-order reaction functions will help clarify how the original conclusions by Jung et al were reached and serve as useful preparation for the complementary higher-order analysis that follows. When the reaction (in this case, between conformational states) and the spatial diffusion are effectively independent, as will be verified for the hairpin molecules, the correlation function in secondorder (also known as order-(1,1)) FCS is given by 45,50,51…”

“…were reached and serve as useful preparation for the complementary higher-order analysis that follows. When the reaction (in this case, between conformational states) and the spatial diffusion are effectively independent, as will be verified for the hairpin molecules, the correlation function in second-order (also known as order-(1,1)) FCS is given by ,, where R 1,1 ( t ) is the “reaction function” which depends only on the reaction and population properties of the involved states. The factors γ 1,1 and Y 1,1 ( t ) depend only on the spatial illumination and detection profiles, collectively called the molecular detection function, and the diffusional properties of the molecules.…”

Sharp, localized phase transitions in single neuronal cells

“…The heating bath was programmed to vary the temperature continuously between two fixed temperatures, with a gradient of ∼0.3 °C/min. Every 1 to 2 min a data point was measured over 15 s. Low laser intensities (10 μW) were chosen to ensure the viability of the cells ( 33 – 35 ). In between, cross-sectional scans were repeatedly recorded to ensure the location of the measurement spot.…”

“…Every 1-2 minutes a data point is measured over 15s. Low laser intensities (10 μW) were chosen to ensure the viability of the cells (33)(34)(35). In between, cross-sectional scans were repeatedly recorded to assure the location of the measurement spot.…”

Imaging of intermittent lipid-receptor interactions reflects changes in live cell membranes upon agonist-receptor binding

“…Like FCS, TRAST measurements combine the sensitivity of fluorescence detection with the environmental sensitivity of the long-lived triplet and other dark transient states that can be monitored. In contrast to FCS however, no particular time resolution in the fluorescence detection is required, and since TRAST measurements do not require single-molecule detection conditions, they can be applied to monitor long-lived dark transient states of fluorescent molecules in a wide range of biological samples 29,30 .…”