A methodology, fluorescence-intensity distribution analysis, hasbeen developed for confocal microscopy studies in which thefluorescence intensity of a sample with a heterogeneous brightnessprofile is monitored. An adjustable formula, modeling the spatialbrightness distribution, and the technique of generating functions forcalculation of theoretical photon count number distributions serve asthe two cornerstones of the methodology. The method permits thesimultaneous determination of concentrations and specific brightnessvalues of a number of individual fluorescent species in solution.Accordingly, we present an extremely sensitive tool to monitor theinteraction of fluorescently labeled molecules or other microparticleswith their respective biological counterparts that should find a wideapplication in life sciences, medicine, and drug discovery. Itspotential is demonstrated by studying the hybridization of5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementaryoligonucleotides and the subsequent cleavage of the DNA hybrids byrestriction enzymes.
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