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Development of Method for the Observation of Light Microscopic Histo-pathological Preparates utilizing Back-scattered Electron Image of Scanning Electron Microscope.

开发利用扫描电子显微镜背散射电子图像观察光显微组织病理学制剂的方法。

基本信息

批准号：
59870018
负责人：
NAKANE Kazuo
金额：
$ 2.5万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1984
资助国家：
日本
起止时间：
1984 至 1985
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-59870018/
关键词：
scanning electron microscope backscattered electron image 免疫組織化学

项目摘要

During the past two decades, electron-immunohistochemical methods have been improved and are indispensable tool for the pathological diagnosis. However those methods are cumbersome and require specialized instruments. Consequently, they have not been used in routine pathology laboratories. In order to make them practical, we investigated the possibility of observing the light microscopic preparations by backscattered electron image (BEI) formed by scanning electron microscope (SEM). JEOL T200 SEM was modified for this purpose. For the observation by BEI, the tissue sections on light microscopic slide must be grounded, since the slide insulate the electrons and result in electron charging at the point of electron impact. Among various organic and inorganic materials examined, ferric ammonium sulfate (FAS) was found to be most practical for the prevention of electron charging. To gain contrast at the sites of antigens, the tissue sections require staining with heavy metals. We found that a repeated exposure to osmium and thiocarbohydrazide was best suited for this purpose. A procedure for the localization of antigen by BEI is summarized;A. One to 2 microns section of fixed tissues embedded in synthetic resin is placed on glass slide and is reacted with horseradish peroxidase conjugated antibody. The peroxidase is then localized using diaminobenzidine and H202.B. The reaction products is chelated with first with osmium, then with thiocarbohydrazide and again with osmium. The section is finally counter-stained with uranyl acetate and treated with FAS.Although with the current instrument, the resolution of the BEI was not as good as that of transmission electron microscope, we were able to localize antigens with the above method with a resolution which is unattainable with the ordinary light microscope. We expect the resolution should be improved as more modifications are made on the instrument and with the method of staining.

在过去的二十年里，电子免疫组织化学方法得到了改进，成为病理诊断不可缺少的工具。然而，这些方法很繁琐，需要专门的工具。因此，它们还没有在常规的病理实验室中使用。为了使其实用化，我们探索了用扫描电子显微镜(SEM)形成的背散射电子图像(BEI)观察光镜制剂的可能性。为此，对JEOL T200扫描电子显微镜进行了改进。对于BEI的观察，光显微镜载玻片上的组织切片必须接地，因为载玻片隔离了电子，并在电子撞击点导致电子带电。在所研究的各种有机和无机材料中，硫酸铁铵(FAS)在防止电子充电方面被发现是最实用的。为了在抗原部位获得对比度，组织切片需要用重金属染色。我们发现，反复暴露在Os和硫代碳肼中最适合于此目的。本文总结了一种BEI定位抗原的方法：A.将1~2微米的固定组织包埋在合成树脂上，放在玻片上，与辣根过氧化物酶标记抗体反应。然后用二氨基联胺和H202.B对过氧化物酶进行定位。反应产物首先与Os、然后与硫代氨基肼、再与Os进行络合。切片最后用醋酸铀酰反染色，FAS处理。虽然目前的仪器BEI的分辨率不如透射电子显微镜，但我们能够用上述方法定位抗原，其分辨率是普通光学显微镜无法达到的。我们预计，随着仪器和染色方法的更多修改，分辨率应该会提高。