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

激光共聚焦扫描显微镜（CLSM）在细胞生物学领域的应用，通过与各种细胞化学技术的结合，在观察细胞功能状态方面取得了显著进展。这一观察不仅证实了生物活性物质在几乎超微结构水平上的定位，而且还赶上了它们的三维构型。最近，CLSM的应用已经扩展到观察活细胞，以观察细胞随时间变化的形态和功能变化。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，在观察生物活性物质的细胞内定位时，对亚细胞器构型的清晰度仍远不如电子显微镜。这种劣势很大程度上可能是由于有证据表明，CLSM获得的“光学（层析）切片”（超过300nm）的厚度仍然远远大于电子显微镜（小于100nm）的厚度。在本研究中，我们打算开发和构建一种新的CLSM，它可以解决这些问题，并能处理对活细胞非常精细结构的观察。为此，我们选择了JEOL Liosonic co . ltd .生产的CLSM （JDLM-6602）作为原型，该原型专门设计用于观察半导体非常精细的表面结构，特别是在垂直轴上获得出色的分辨率，并且使“针孔”更小。为了提高分辨率，我们在外部安装了He-Cd激光辐射单元（波长短至325nm）。我们还应用了高性能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.