PHARMCOLOGICAL STUDY ON THE 3-DIMENSIONAL ULTRASTRUCTURE OF A BLOOD PLATELET

为了用高压电子显微镜（HVEM）观察血小板的三维超微结构，我们主要采用临界点干燥法制备样品，其重现性不尽如人意。在本研究中，我们采用冷冻干燥法建立了该方法，该方法不包括与有机溶剂和液态二氧化碳的介质交换步骤。因此，以戊二醛为前缀的血小板被附着在涂有聚l赖氨酸的聚乙烯正规网片上。用戊二醛和四氧化锇后固定，并用醋酸铀酰染色。用液氮快速冷冻后，在组织冷冻干燥机（EMSCOPE FD-500）中干燥，用HVEM （HITACHI, H-1250 M）观察，并与常规电镜超薄切片法观察结果进行比较。致密体、5 -羟色胺储存位点以及微管的周向带被清晰地观察到。我们还可以识别α颗粒和线粒体，其特征外观有些模糊。然而，未发现糖原颗粒。利用立体配对图像对细胞内小管系统和开放小管系统进行三维分析。我们还分析了各种药理学修饰的影响，包括血清素、冷应激和一些细胞表面示踪剂如钌红。HVEM的工作是在国家生理科学研究所（冈崎，日本）的联合研究计划下完成的。

In order to examine the 3-dimensional ultrastructure of a blood platelet with a high voltage electron microscope (HVEM), we had been mainly prepared the sample by critical point drying method, which give us somewhat unsatisfactory results as to its reproducibility. In this study, we established the method using freeze drying method, which did not include the medium exchange steps with organic solvent and liquid carbon dioxide. Thus, platelets, prefixed with glutaraldehyde, were attached to polyvinylformal sheet-mesh which had been coated with poly-L- lysine. They were post-fixed with glutaraldehyde and osmium tetroxide, and were stained with uranyl acetate. After rapid freezing with liquid nitrogen, they were dried in a tissue freeze dryer (EMSCOPE FD-500), and observation was made with a HVEM (HITACHI, H-1250 M), results of which were compared with that had been obtained by the ultra-thin sectioning method using a conventional electron microscope. Dense bodies, serotonin storage sites, was clearly observed as well as circumferential bands of microtubules. We could also identify alpha-granules and mitochondria, the characteristic appearance of which was somewhat obscure. Glycogen granules, however, were not identified. Furthermore, intracellular canalicular system and open canalicular system were 3-dimensionally analyzed by the stereo- paired images. We also analyzed the effect of various pharmacological modification, including serotonin, cold stress and some cell surface tracers such as ruthenium red. The work with a HVEM was done under the joint study program of National Institute for Physiological Sciences (Okazaki, Japan).