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

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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.