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

我们制作了一个显微镜，其中两个激发波长交替变化。图像被数字化并存储在微型计算机中。该仪器与可报告膜电位和离子浓度等各种数量的荧光染料相结合，使这些数量的同时测量变得可行，并有助于了解细胞功能的机制。(1)建立了用二氢霍达明123检测胞内H_2O_2生成的方法。用该方法结合显微镜，测定了表皮角质形成细胞中H_2O_2形成的紫外作用谱。(2)目前输血的问题之一是防止紫外线照射引起的异敏反应。然而，其机制尚不清楚。我们在显微镜下测量了紫外线照射下各种血细胞的活性氧形成，以及细胞内钙浓度和膜电位的变化。(3)真核细胞的膜电位不能用正变探针测量，因为它在线粒体中有大量的积累。我们设计了校正方法并应用于肝细胞膜电位的测定。(4)使用带负电荷的荧光染料（merocyanine 540），我们无法测量肝细胞的膜电位，提示有必要进一步研究染料与细胞膜的相互作用。

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.