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Particle counting by fluorescence correlation spectroscopy. Simultaneous measurement of aggregation and diffusion of
1Department of Biophysical Chemistry, Biocenter of the University of Basel, Switzerland.
Abstract:
A method for simultaneous determination of molar weights (M) and lateral diffusion constants (D) of particles in three- and two-dimensional systems is described. Spontaneous concentration fluctuations in space and time are analyzed, by monitoring fluctuations in the fluorescence from fluorescein-labeled molecules (1 dye/molecule is sufficient), excited by a rotating laser spot. For particles in solution, M values are determined over the range of 3 x 10(2) to 3 x 10(11) daltons, and D values can be determined from approximately 10(-7) to 10(-10) cm2/s. The time for a determination is approximately 1 min. Aggregation can be followed by changes of either M or D. This method is used to study the calcium dependence of vesicle aggregation or fusion, and the time course of aggregate formation of porin (an Escherichia Coli outer membrane protein) in lipid monolayers. Essential parameters for the development of the method are described. Equations to estimate the signal-to-noise ratios and to find the optimal free parameters for a specific application are derived. The theoretical predictions for the correlation function of the signal and for the signal-to-noise ratio are compared with observed values.