Total Internal Reflection-Fluorescence Correlation Spectroscopy

Abstract

The combination of total internal reflection illumination with fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique. This method allows measurement of at least three key properties of fluorophores very close to surface/solution interfaces, including the local fluorophore concentration, the local fluorophore translational mobility, and the kinetic rate constants which describe the reversible association of fluorophores with the interface. This review describes the conceptual basis of TIR-FCS, aspects important to the experimental realization of this technique, and methods for theoretically modeling and analyzing data. Previous experimental applications of TIR-FCS are also summarized, including studies of protein dynamics very close to substrate-supported planar membranes, measurement of the kinetic dissociation rate for fluorescent ligands specifically and reversibly associating with receptors in substrate-supported planar membranes, the interaction of small dye molecules with chromatographic surfaces, investigations of fluorophore behavior in sol-gel films, and the monitoring of intracellular vesicular motion near the inner leaflet of plasma membranes in intact, live cells. A number of potential future directions for TIR-FCS are indicated, for example, the use of very high refractive index substrates or small, metallic substrate-deposited structures, the use of photon counting histograms, the use of a single fluorescent reporter molecule to provide kinetic rate constants for nonfluorescent molecules which compete for the same surface sites, and more sophisticated methods of data analysis including two-color cross-correlation and high-order autocorrelation.