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

本课题将普通激光共聚焦显微镜的特征扩展到近紫外波长。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。我们改进了显微镜本体和物镜的光学结构，并成功地研制出一种显微镜系统，它可以利用从可见光区到近紫外区的广泛波长的光来成像。结合贴片夹持实验，我们进一步开发了控制共聚焦显微镜的软件。利用该共聚焦显微镜系统，我们可以用双波长荧光指示剂indo-1显示细胞内Ca浓度。实时分割吲哚-1荧光，制作反映细胞内钙浓度的比值图像。我们已经开发了一种特殊的激光扫描方法，在这里被称为飞带扫描。通过这种FBS扫描方法，我们可以观察到高时间分辨率和几个限制区域的高特殊分辨率。FBS可以与笼化化合物的活化相结合，例如，笼化Ca的释放可以通过时间推移的方式进行监测。FBS扫描积累的图像数据通过网络传输到实验室的另一台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，特别是用于在样品的限制区域激活笼状化合物。作为一个整体系统，我们所开发的共聚焦显微镜可以结合成像、笼状化合物的激活和利用贴片电极的电生理研究。

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.