Application of a UV-laser to a confocal microscope and the observation of intracellular Ca2+dynamics.

本研究将普通激光共聚焦显微镜的功能扩展到近紫外波段。然后，我们将扩展的紫外激光共聚焦显微镜应用于细胞生理学的研究。我们改进了显微镜体和物镜透镜的光学系统，并成功地开发了一种显微镜系统，该系统可以利用从可见光区到近紫外区的宽波长范围的光成像。结合膜片钳实验，进一步开发了共聚焦显微镜控制软件。通过利用这种共聚焦显微镜系统，我们可以使双波长荧光指示剂indo-1指示的细胞内钙浓度的图像。对Indo-1荧光进行真实的时间分割，并制作反映细胞内Ca浓度的比率图像。我们开发了一种特殊的激光扫描方法，在这里称为飞带扫描。利用这种FBS扫描方法，我们可以对几个有限区域进行高时间分辨率和高空间分辨率的观测。FBS可以与笼状化合物的活化组合，并且例如进行笼状Ca的释放并以延时方式监测。FBS扫描积累的图像数据通过网络传输到实验室的其他工作站进行进一步分析。我们还开发了一种特殊的激光扫描方法，特别是在样品的限制区域的笼状化合物的激活。作为一个完整的系统，我们已经开发的共聚焦显微镜可以结合联合收割机成像，与激活的笼化合物和电生理研究利用贴片电极。

Feature of an ordinary confocal laser microscope was extended in this research project upto the wavelength of near Ultraviolet. We have then applied the extended UV laser confocal microscope into the investigation of cellular physiology. We have modified the optics of the microscope body and the objective lens and have succeeded in developing a microsope system which can make images utilizing a wide range of wavelength light from the visible region to near ultraviolet region. We have further developed a software to control the confocal microscope in conbination with the experiment of patch clamping. By utilizing this confocal microscope system we could make images of intracellular Ca concentration indicated by the dual wavelength fluorescence indicator, indo-1. Indo-1 fluorescences were real time divided and ratio images were made which were reflecting the intracellular Ca concentration. We have developed a special laser scanning method called here as a Flying Band Scan. By this FBS scanning method, we could make observation of high time resolution with high spetial resolution of several restricted regions. The FBS could be combined with the activation of a caged compound, and the release of caged Ca, for example, was made and was monitored in a time lapse manner. The image data accumulated by FBS scanning was transmitted to the other workstation in the laboratory through network for further analysis. We also developed a special laser scanning method especially for the activation of a caged compound at a restricted region of the specimen. As a total system, the confocal microscope we have developed could combine imaging, with the activation of a caged coumpound and with the electrophysiological investigation utilizing a patch electrode.