Measurements of diffusion and chemical kinetics by fluorescence photobleaching recovery and fluorescence correlation spectroscopy

N. O. Petersen, E. L. Elson

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41 Scopus citations

Abstract

Chemical kinetics and rates of lateral transport (diffusion or flow) can be measured by observing spontaneous fluctuations in reactant concentrations which occur even when the system rests in equilibrium. This can be accomplished by using fluorescence to monitor the numbers of molecules of specified types in a defined open region of the reaction system. The feasibility of this approach has been demonstrated, and the method, called fluorescence correlation spectroscopy (FCS) has found practical application. Because it is based on microscopic fluctuations FCS is an intrinsically statistical method, however, and so requires that the reaction system be stable during prolonged measuring intervals. For studies of less stable systems (e.g., living cells in culture) a method has been developed which is based on observation of the rate of relaxation of a local macroscopic concentration gradient. The gradient is produced by rapidly photolyzing a portion of the reactant molecules in a small open region of the system. This method, which we call fluorescence photobleaching recovery (FPR), yields results which are approximately equivalent to those obtained by FCS. Theoretical advantages of FCS are balanced by practical experimental advantages of FPR. The major applications of this combined technology up to now have been in studies of lateral diffusion in model membranes (FCS and FPR) and on animal cell surfaces (FPR).

Original languageEnglish
Pages (from-to)454-484
Number of pages31
JournalMethods in enzymology
Volume130
Issue numberC
DOIs
StatePublished - Jan 1 1986

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