Abstract
A direct method for the microfluorimetric determination of NADPH2 and 4-methylumbelliferon in the sub-picomole range is described. By means of an inverted fluorescence microscope with epi-illumination it has been possible to measure low concentrations of these fluorophores in volumes down to 17 nl. The aqueous microvolumes to be measured are suspended in oil to prevent evaporation. Since the droplets are sunk on thin teflon foil, to attain a spherical form, they can be measured in a reproducible way. The excitation of the microdroplet takes place from below through the teflon foil, while the fluorescence emission follows the same route back to a photomultiplier. The possibilities of this method are illustrated with the biochemical measurements of glucose-6-phosphate dehydrogenase (G6PD) activity in single fibroblasts. It was possible to distinguish between normal and G6PD deficient cells on a single cell level. The microfluorimetric measurement of lysosomal enzymes with 4-methylumbelliferyl substrates is discussed.
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