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

在过去的二十年中，电子免疫组织化学方法得到了改进，是病理诊断不可或缺的工具。然而，这些方法很麻烦，需要专门的仪器。因此，它们尚未在常规病理实验室中使用。为了使其实现，我们研究了利用扫描电子显微镜（SEM）形成的背散射电子图像（BEI）来观察光显微制备的可能性。为此对JEOL T200 SEM进行了修改。对于BEI观察，光学显微镜载玻片上的组织切片必须接地，因为载玻片使电子绝缘，导致电子在电子撞击点带电。在各种有机和无机材料中，硫酸铁铵（FAS）被认为是最实用的防止电子充电的材料。为了在抗原位置获得对比，组织切片需要用重金属染色。我们发现反复暴露于锇和硫代碳肼是最适合这个目的的。本文综述了用BEI定位抗原的方法。将一至2微米的固定组织切片包埋在合成树脂中，置于玻片上，与辣根过氧化物酶偶联抗体反应。然后用二氨基联苯胺和h2 . b定位过氧化物酶。反应产物首先与锇，然后与硫代碳酰肼，再与锇进行螯合。切片最后用醋酸铀酰反染，并用FAS处理。虽然目前的仪器，BEI的分辨率不如透射电子显微镜，但我们可以用上述方法定位抗原，分辨率是普通光学显微镜无法达到的。我们期望随着仪器和染色方法的改进，分辨率会有所提高。

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.