Standardization and Quality Assurance in Fluorescence Measurements II

Abstract

This chapter presents a review of fluorescence correlation spectroscopy (FCS), an experimental technique with single-molecule sensitivity, which is based on the analysis of fluctuations of fluorescence intensity detected from a tiny volume. Correlation functions of fluorescence fluctuations can provide information on the translational and rotational diffusion of fluorophores, dynamics of singlet–triplet transitions, chemical reactions, flow, and active transport of fluorescent molecules. A detailed theoretical description of the fluorescence correlation and cross-correlation technique is followed by a discussion of various experimental aspects of FCS, including the choice of instrumentation and fluorophores, sample- and setup-related nonidealities and possible artifacts, statistical accuracy, and approaches to the analysis of FCS data. Additionally, some FCS application aspects are addressed, including the quantitative determination of translational diffusion coefficients and the use of FCS in studies of (bio)polymers and phospholipid membranes. The chapter concludes with an overview of both well-established and currently emerging varieties of FCS and related methods, including the use of two-photon excitation and the application of total internal reflection, nanoapertures, and stimulated emission depletion to confine the detection volume; the use of higher-order correlations; application of time-resolved and time-gated detection; multifocal and CCD-based FCS; and image correlation and scanning FCS techniques.