Interpretation of Fluorescence Correlation Spectroscopy and Photobleaching Recovery in Terms of Molecular Interactions

Fluorescence photobleaching recovery (FPR) and fluorescence correlation spectroscopy (FCS) are two closely related methods for measuring the rates of diffusion, flow, and chemical reactions. Although the two methods share similar theoretical bases and experimental methods, they differ in that FPR measures the dissipation of a macroscopic concentration gradient generated by pulse photolysis, whereas FCS measures the average time course of spontaneous microscopic concentration fluctuations that are constantly occurring even though the system is in equilibrium. Both FPR and FCS are based on the measurements of the fluorescence emitted from a small open observation subregion of the sample. The measured intensity varies with the number of fluorescent molecules and so increases or decreases as these molecules enter or leave the subregion. Hence, knowing the size of the observation region, diffusion coefficients and drift or flow velocities can be determined from the measurements of the rate of change of fluorescence reactants. Hence, FCS and FPR can be used to characterize both transport properties and chemical reaction kinetics. This chapter discusses the conceptual bases, theory, capabilities, and interpretation of these two approaches, emphasizing their application to measure molecular interactions.