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，电子免疫组化方法得到了改进，并成为病理诊断不可缺少的工具。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学实验室中使用。为了使它们实用，我们研究了通过扫描电子显微镜（SEM）形成的背散射电子图像（BEI）观察光显微制剂的可能性。为此，对JEOL T200 SEM进行了改进。对于BEI观察，光学显微镜载玻片上的组织切片必须接地，因为载玻片绝缘电子并导致电子撞击点处的电子充电。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。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，反复暴露于锇和硫代碳酰肼最适合于此目的。总结了BEI定位抗原的方法;将包埋在合成树脂中的1 - 2微米固定组织切片置于载玻片上，并与辣根过氧化物酶结合抗体反应。然后使用二氨基联苯胺和H2 O2定位过氧化物酶。反应产物首先与锇螯合，然后与硫代碳酰肼螯合，再与锇螯合。用醋酸双氧铀复染后，用FAS处理。虽然用目前的仪器，BEI的分辨率不如透射电镜，但我们能用上述方法定位抗原，其分辨率是普通光学显微镜所不能达到的。我们希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.