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

点击翻译按钮获取中文摘要

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.