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Development of molecular anatomical technique of the cell by the scanning probe microscop

扫描探针显微镜细胞分子解剖技术的发展

基本信息

批准号：
07557003
负责人：
YAMASHINA Shohei
金额：
$ 10.18万
依托单位：
Kitasato University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

Cellular ultra stucture and identification of functional molecules was determined with a use of the present SPM,and following results were obtained.(1) Uitra fine structure of cells as revealed by SPMComparative study of various sample preparation imdicated that the best resolution could be obtained by a combination of cyclic contact mode with ultra sharpen needle. According to this, cellular ultra structure was resolved at about 30nm resolution. Further sharpening of needle tip was feasible in order to power up the resolution.(2) Application of freeze fractured and SDS treated sample.Observation of freeze fractured and SDS treated samples were made to determine the true surface of the cell under the best condition stated above. As a result, irregular processes about 30nm in diameter could be visualized on the cellular true surface. These structure seemed to correspond with compounds of lipid and protein molecules that consisted of cellular membrane. It was expected to visualize more detailed image of the cell surface by this preparation, if resolution of the SPM could be raised.(3) Identification of functional molecules by the SPMColloidal gold particle labelled by immunohistochemical method could be detected by their configuration, however, lateral diameter tended to appear slightly larger than expected amount due to lateral effect of needle tip. Not only gold particles but also lead phosphate, final reaction products of enzyme histochemical reaction could be detected with a use KFM that made it possible to detect the difference in surface electrical potential. The present KFM has several problems of slow response to local potential differences.

利用本扫描显微镜对细胞超微结构和功能分子进行了鉴定，得到了以下结果：(1)扫描电子显微镜揭示了细胞的超精细结构。不同样品制备方法的比较研究表明，采用循环接触模式和超锐针相结合的方法可以获得最佳的分辨率。在此基础上，对细胞超微结构进行了约30 nm的分辨。进一步的针尖锐化是可行的，以提高分辨率。(2)冷冻断裂和十二烷基硫酸钠处理的样品的应用。在上述最佳条件下，对冷冻断裂和十二烷基硫酸钠处理的样品进行观察，以确定细胞的真实表面。结果，细胞真表面可见直径约30 nm的不规则突起。这些结构似乎与组成细胞膜的脂类和蛋白质分子的化合物相对应。(3)免疫组织化学方法标记的胶体金颗粒可以通过其形态检测到功能分子，但由于针尖的侧向作用，其横径往往比预期值略大。不仅可以检测到金粒子，而且可以检测到酶组织化学反应的最终反应产物磷酸铅，这使得检测表面电位的差异成为可能。目前的KFM存在对当地潜在差异反应迟缓的几个问题。