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Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Photobleaching Recovery (FPR) are closely related technically but have an important difference in principle. The former extracts information from measurements of spontaneous concentration fluctuations, the latter, from measurements of the relaxation of a concentration gradient produced by a macroscopic perturbation. By monitoring fluorescence changes in an open region of the reaction system both methods can determine rates of molecular transport. Because of the high molecular specificity possible with fluorescence labels, the high spatial resolution attainable with laser-microscope excitation, and the relatively nonperturbing nature of the measurements, these methods have many potential uses. Although the two methods are fundamentally equivalent, FCS is simpler in concept and in analysis, but can be applied only to relatively stable systems. Typically the open observation region is defined by a laser beam which excites the measured fluorescence and is focused and detected using confocal microscope optics. Most previous applications have been to two-dimensional membrane systems. Although a number of interesting measurements have also been carried out on three-dimensional bulk solution systems, additional complications arise due to the variation in intensity and radius of the focused laser beam along the optic axis (z-axis). Because of the former the motion of particles in the z direction can be detected. Because of the latter the characteristic diffusion time, defined by the beam radius, differs for particles at different distances from the focal plane. Usually the latter is the dominant effect as confirmed both by theory and experimental measurements. An additional but essential complication arises from placing a field diaphragm before the photodetector to reduce the off-focus background signal. A natural extension of FCS is Fluorescence Distribution Sepctroscopy (FDS) which provides information about the distribution of aggregate sizes in systems of fluorescent molecules. This information is obtained from an analysis of the distribution of fluorescence photocounts emitted in a series of defined time intervals. The nonperturbing nature of FDS makes it especially useful for the analysis of reversible aggregation processes often found in biological systems. FDS measurements in three-dimensional systems are especially sensitive to variations in beam intensity and radius along the z direction.
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