Fluorescence Correlation Spectroscopy. I. Conceptual Basis and Theory英文文献.pdfVIP

BIOPOLY MERS VOL. 13, 1-27 (1974) Fluorescence Correlation Spectroscopy. I. Conceptual Basis and Theory ELLIOT L. ELSON, Department of Chemistry, Cornell University, Ithaca, New York 14850and DOUGLAS MAGDE,* Department of Applied Physics, Cornell University, Ithaca, New York 14850 synopsis We describe a method for determining chemical kinetic constants and diffusion coeffi- cients by measuring the rates of decay of spontaneous concentration fluctuations. The equilibrium of the system is not disturbed during the measurement. We measure the number of molecules of a specified type in a defined open volume as a function of time and compute the time course of the deviations from the thermodynamic mean concen- tration. The method is based on the principle that the rates of decay of spontaneous microscopic fluctuations are determined by the same phenomenological rate coefficients as those of macroscopic departures from equilibrium which result from external per- turbations. Hence, an analysis of fluctuations yields the same chemical rate constants and diffusion coefficients as are measured by conventional procedures. In practice the number of the specified molecules is measured by a property such as absorbance or fluorescence which is specific and sensitive to chemical change. The sample volume is defined by a light beam which traverses the cell. As the molecules appear in or disap pear from the light beam, either due to diffusion or chemical reaction, their concentra- tion fluctuations give rise to corresponding fluctuations of the intensity of absorbed or emitted light. This paper presents the theory needed to der