| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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).
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