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Surface density of radiant sources measured by optical microscopy : correction for diffraction and focus limitations Knowles, David William


A new technique is introduced for the determination of the surface density of membrane bound components on living cells inuitro. This technique provides a simple conversion of fluorescence intensity to number density and involved the modelling of the spatial optical response of the microphotometer to account for the inherent diffraction and focus limitations of the system. A theoretical and experimental study was undertaken to examine the adsorption of a fluorescently labelled ligand (WGA) onto the membrane surface of a biological cell (the erythrocyte). WG A/red cell interaction was evaluated with a fluorescence microphometer. The microphotometer is a laser based fluorescence microscope in combination with intensified video imaging and digitizing equipment that produces fluorescence images with a resolution of 0.25μm. The fluorescence conversion technique was used to characterize the adsorption of WGA on to the red cell surface. Individual cells were isolated and incubated in various bulk concentrations of fluorescenated WGA to determine the dependence of adsorbed concentration on bulk concentration and incubation time. The equilibrium results gave a microscopic association constant of 2.95x10⁸ liters/mole, a molecular binding ratio of one WGA molecule per glycophorin molecule on the red cell surface and the number of glycophorin molecules per human red blood cell as (6.5±0.3)x10⁵.

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