Brief introduction to fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) measures rates of transport (diffusion coefficients, convection velocities) and chemical reactions (rate constants) in equilibrium or nonequilibrium steady-state systems without the need for a perturbation of the state of the system. The rates are extracted from a record of fluorescence fluctuations observed from an open laser-illuminated observation volume. In addition, FCS provides the number of measured fluorescent molecules in the observation volume and, therefore, their molecular brightness. This enables sensitive measurements of aggregation of the fluorescent system components. FCS is now used as a routine tool for studying systems in physics, chemistry, and biology. Recent extensions of the fluorescence fluctuation approach have included methods based on imaging and cross correlation. Because of the sensitivity and chemical specificity of fluorescence detection and also the ability to form a very small diffraction-limited detection volume, FCS is especially useful for studies of dynamic molecular processes in living biological cells. FCS provides a window on mesoscopic systems and is closely related to fluorescence methods for studying single molecules.