Fluorescence Correlation Spectroscopy (FCS) is a tool that provides quantitative localized measurements of important physical parameters including mechanisms of transport, molecular mobilities, and densities of fluorescently labeled species. In the most basic configuration, FCS examines the inherent correlations exhibited by the fluctuating fluorescent signal from labeled molecules as they transition into and out of a specified excitation volume or area. The dual-color variation, termed Fluorescence Cross-Correlation Spectroscopy (FCCS), is utilized to probe two species labeled with different fluorophores. FCCS can extend investigations to the examination of biochemical reactions between two partners, such as reaction rates, kinetics, fractions of binding or reacting molecules, and mobilities of a complex formed between the partners.
Bacia, K., Kim, S. A. and Schwille, P.
Fluorescence cross-correlation spectroscopy in living cells. Nature Methods 3: 83-89 (2006). An excellent introduction to the use of FCS and cross-correlation (FCCS) techniques to examine a host of processes on the molecular scale, including diffusion, binding, enzymatic reactions, and co-diffusion of two different species. FCCS is described in the context of FRET and multicolor imaging.
Krichevsky, O. and Bonnet, G.
Fluorescence correlation spectroscopy: The technique and its applications. Reports on Progress in Physics 65: 251-297 (2002). A comprehensive review of fluorescence correlation spectroscopy techniques both from a practical and theoretical perspective. Reviewed are experimental design and setup, recent applications in analytical chemistry, cell biology, and physics, as well as application of the technique using laser scanning confocal techniques.
Tcherniak, A., Reznik, C., Link, S. and Landes, C. F.
Fluorescence correlation spectroscopy: Criteria for analysis in complex systems. Analytical Chemistry 81: 746-754 (2009). The authors evaluate the effect of varying three key parameters in FCS analysis, including minimum and maximum lag time, averaging times, and demonstrate that use of appropriate settings plays a critical role in recovering accurate and reliable decay times and resulting diffusion constants. The article contains many helpful references.
Bacia K. and Schwille, P.
A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. Methods 29: 74-85 (2003). An excellent general review of FCS aimed at investigations using live-cell imaging. The article provides an overview of recent applications and a practical guide for new investigators who are seeking to integrate FCS into live-cell imaging to obtain information on particle mobility. This review article also emphasizes instrument configuration, fluorophore choice, and positioning of the measurement volume.
Digman, M. A. and Gratton, E.
Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. Wiley Interdisciplinary Reviews: Systems Biology and Medicine 1: 273-282 (2009). A quantitative review of the underlying statistical assumptions of image correlation spectroscopy analysis, including simulations used for illustrative purposes, rather than a discussion of applications. The article focuses on methods based on fluctuation correlation spectroscopy to determine the formation of protein complexes in living cells.
Altan-Bonnet, N. and Altan-Bonnet, G.
Fluorescence correlation spectroscopy in living cells: A practical approach. Current Protocols in Cell Biology 4.24: 24.1-24.14 (2009). A practical guide to fluorescence correlation spectroscopy as applied to living cells expressing fluorescently labeled proteins and lipids to determine the diffusion timescales and the total number of diffusing fluorescent molecules in the cell. The article includes a step-by-step approach to performing FCS that covers instrument configuration, data acquisition, and analysis.
Haustein, E. and Schwille, P.
Fluorescence correlation spectroscopy: Novel variations of an established technique. Annual review of Biophysics and Biomolecular Structure 36: 151-169 (2007). The authors present an excellent review with superb illustrations and a comprehensive reference section. Discussed are the basic aspects of FCS instrumentation and analysis, cross-correlation methodology, and alternative detection schemes. This review article is highly recommended for investigators new to the technique.
Petrasek, Z. and Schwille, P.
Fluctuations as a source of information in fluorescence microscopy. Journal of the Royal Society Interface 6: S15-S25 (2009). Examination of fluorescence correlation spectroscopy in terms of obtaining more quantitative information from the data than that given by average values, while minimizing the effects of noise due to stochastic photon detection. The article includes a general review of single-point measurements and imaging techniques.
Weiss, M.
Probing the interior of living cells with fluorescence correlation spectroscopy. Annals of the New York Academy of Sciences 1130: 21-27 (2008). Beginning with a primer on diffusion, this excellent review article first covers the basic aspects of FCS, and then advances to material properties, testing the crowded state of intracellular fluids, and finally covers the generic consequences of anomalous diffusion in viscoelastic fluids.
Haustein E. and Schwille, P.
Single-molecule spectroscopic methods. Current Opinion in Structural Biology 14: 531-540 (2004). The authors review the single-molecule-sensitive technique of fluorescence correlation spectroscopy to enable real-time access to a multitude of molecular parameters, including diffusion coefficients, concentration, and molecular interactions. Also discussed is the localization of fluorophores on nanometer length scales.