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Free keywords:
Luminescence; Proteins; Laser scanners; Microscopy; Microscopes; Diffusion
Abstract:
Laser scanning microscopy was used to study dynamic processes in single cells. The laser scanning microscope of Heidelberg Instruments was complemented with a 1W krypton laser and a microinjection set-up. Simple algorithms were worked out which permit determination of local and integrated intensities in fluorescence scans. In one application the lateral diffusion of macromolecules was studied. The krypton laser was used to irreversibly photolyse fluorescently labeled dextrans in small volumes of a thin fluid layer; equilibration of the local fluorescence inhomogeneity by lateral diffusion was followed by repetitive scanning. In a second application the permeability of single red blood cell membranes which had been exposed to the complement cascade was studied. In a further application artificial nuclear proteins, constructed by molecular genetic methods, were injected into the cytoplasm of hepatoma cells. The kinetics of protein transport from cytoplasm to nucleus were derived from fluorescence scans. In all applications a good agreement between results obtained by laser scanning microscopy and those obtained independently by other methods and instruments was observed.
