Development of Fluorescent Microscope Having Multi-Excitation for Simultaneous Measurements of Several Dyes in Order to Investigate Cell Functions

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We made a microscope where two excitation wavelengths were changed alternatively. The image was digitized and stored in a micro-computer. With combination of this instrument and various fluorescent dyes that can report various quantities such as membrane potential and ionic concentrations, the simultaneous measurement of these quantities becomes feasible and may help the understanding of the mechanism of cell functions.(1) The method to detect intracellular H_2O_2 formation using dihydrorhodamine 123 was developed. With this method together with the microscope, we determined ultra-violet action spectrum for H_2O_2 formation in epidermal keratinocytes.(2) One of current problems in transfusion is to prevent allosensitization by UV irradiation. The mechanism, however, is not clear. We measured active oxygen formation by UV irradiation in various blood cells, as well as change in intracellular Ca concentration and membrane potential with the microscope.(3) One cannot measure the membrane potential of eucaryotic cells with a positively changed probe, because of high accumulation into mitochondria. We devised the correction method and applied to measure the membrane potential of hepatic cells.(4) With a negatively charged fluorescent dye (merocyanine 540), we failed to measure the membrane potential of hepatic cells, suggesting the necessity of further study on the interaction of a dye and cell membrane.