Development of a New High Performance Confocal Laser Scanning Microscpe Enabling the Observation of Dynamic Changes of Subcellular Organelles.

共聚焦激光扫描显微镜（CLSM）在细胞生物学领域的应用，使其与多种细胞化学技术相结合，在观察细胞功能状态方面取得了显著进展。该观察不仅能够在几乎超微结构水平上确认生物活性物质的定位，而且还能捕捉到它们的三维构型。最近，CLSM的应用已经扩展到观察活细胞，以观察细胞的时间依赖性形态和功能变化。然而，我们必须承认，距离这些努力的最终目标还有相当大的距离。例如，在生物活性物质的细胞内定位的观察中，亚细胞器的构型的清晰度仍然远远低于电子显微镜。这种劣势可能主要是由于CLSM中获得的“光学（断层）切片”的厚度（超过300 nm）仍然比电子显微镜（小于100 nm）大得多。在本研究中，我们打算开发和构建一种新的CLSM，它可以处理这些问题，并将科普观察活细胞的非常精细的结构。为此目的，我们选择了一个CLSM（JDLM-6602）生产的JEOL Liosonic Co.Ltd.as的原型，这是专门设计用于观察非常精细的半导体表面结构，通过获得优异的分辨率，特别是在垂直轴上，并通过使“针孔”小得多。为了提高分辨率，我们同时安装了He-Cd激光照射装置（波长短至325 nm）。我们还应用了一个高性能的光子计数器来检测来自样品的非常微弱的信号。

The application of the confocal laser scanning microscope (CLSM) in the filed of cell biology brought about outstanding progress in observing functional states of cells through the combination with various cytochemical techniques. The observation enabled not only to confirm the localizations of biologically active substances at almost ultrastructural levels but also catch up their 3 dimensional configurations. Recently, the CLSM application has been extended to observe living cells to observe the time dependent morphological and functional changes of the cells. We have to admit, however, that the considerable distance has still been left from the ultimate goal of those attempts. For instance, in the observations of intracellular localizations of biologically active substances, the clearities of the configurations of subcellular organelles are still far less than those of electron microscopy. This inferiority may largely be due to the evidence that the thickness of "optical (tomographic) sections" obtained in CLSM (more than 300nm) is still much bigger than that of electron microscopy (less than 100nm). In the present study, we intended to develop and construct a new CLSM which can manage such problems and would cope with the observation of very fine structures of living cells. For this purpose, we selected a CLSM (JDLM-6602) produced by JEOL Liosonic Co.Ltd.as a prototype which was specially designed to observe very fine surface structures of semiconductors by obtaining excellent resolution especially in vertical axis and by making "pin hole" much smaller. We installed extraneously the He-Cd laser radiation unit (the wave length as short as 325nm) to improve the resolution. We also applied a high performance photon counter to detect very weak signals from the specimens.