Fluorescence Correlation Spectroscopy: Molecular Complexing in Solution and in Living Cells

This chapter describes how the microscope can be used to measure a fluorescence signal from a small, confined volume of the sample-the confocal volume-and how these measurements are used to quantitate the dynamics and complexing of molecules, the technique of fluorescence correlation spectroscopy (FCS). FCS represents a significant example of how the microscope can be used to extract information beyond the resolution limit of classical optics. FCS enables studying events at the level of single molecules. With FCS, one can measure the diffusion times and the interaction of macromolecules, the absolute concentration of fluorescently labeled particles, and the kinetics of chemical reactions. Practical applications of FCS include studies on ligand-receptor binding, protein-protein and protein-DNA interactions, and the aggregation of fluorescently labeled particles. The chapter focuses on the principles of FCS, demonstrates how FCS is used to study macromolecular interactions in solution and in living cells, and examines critical experimental parameters that must be considered. The chapter also discusses the minimum requirements for building a microscope-based FCS instrument and illustrates the key criteria for both instrument sensitivity and analysis of FCS data. It can be used to study single molecules both in solution and in living cells and can be used to monitor a variety of macromolecular interactions. When used as an in vitro technique, FCS measurements are easy to conduct and can be made on simplified instrumentation. When used in vivo on living cells, many additional factors must be considered when evaluating experimental data. Despite these concerns, FCS represents a new approach that has broad applicability for the determination of molecular stoichiometry both in vivo and in vitro for a variety of membrane and soluble receptor systems.