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Biophysical Journal 16: 1315-1329 (1976)
© 1976 the Biophysical Society
ABSTRACT
We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.
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