Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) was developed in order to char­ acterize the dynamics of molecular processes in systems in thermodynamic equilibrium. FCS determines transport and chemical reaction rates from mea­ surements of spontaneous microscopic thermally driven molecular concentra­ tion fluctuations. Since its inception, and particularly in recent years, techni­ cal and conceptual advances have extended the range of practical applicability and the information obtainable from FCS measurements. Improvements in microscopy, data acquisition, and data processing have greatly shortened the time required for FCS measurements. FCS can now be routinely applied to labile systems such as cells, and for the acquisition of large volumes of data as required for high-throughput screening. Cross correlation methods pro­ vide a powerful tool for characterizing interactions among different molecular species. Analysis of the amplitude of concentration fluctuations can provide a wealth of information about aggregation/polymerization process and the compositions of mixtures. Furthermore, FCS provides a bridge between conventional measurements of dynamic processes on a macroscopic concentration scale and the currently developing field of single molecule measurements. Both FCS and single mole­ cule approaches measure directly stochastic fluctuations in molecular pro­ perties, and so must be analyzed by statistical methods to yield conventional phenomenological parameters. As commonly practiced, FCS yields these phe­ nomenological parameters, e. g. , diffusion coefficients and chemical rate con­ stants, directly in terms of a fluorescence fluctuation autocorrelation func­ tion.

This book presents the theoretical background to fluorescence correlation spectroscopy (FCS) and a variety of applications in various fields of science. FCS is based on the detection of single molecules excited to fluorescence in diffraction limited confocal volume elements and the time correlation of stochastic events. It provides ultimate sensitivity in the analysis of molecular processes and has found numerous applications in physics, chemistry and particularly in biomolecular sciences. Its high spatial and temporal resolution has made FCS a powerful tool for the analysis of molecular interactions and kinetics, transport properties due to thermal motion and flow, as well as the physics of the excited state in solution as well as at the cellular level. Its application in high throughput drug screening is using all the potential of this prime analytical tool.