Mizuhira V, Hasegawa H, Notoya M
Department of Cell Biology and Anatomy, Medical Research Institute, Tokyo Medical and Dental University, Japan.
Prog Histochem Cytochem. 2000;35(2):67-183.
We tested various fixation and analysis methods to demonstrate by electron microscopy elemental imaging in tissues and cells, i.e., soluble substances such as many kinds of ionic elements, water soluble low molecular peptides, and even organic solvent soluble substances such as lipids. For the ionic elements, we tested frozen dried or freeze-substituted methods and organic or inorganic special chemical precipitation methods combined with microwaved fixation methods. The data were analyzed with electron beam X-ray microanalysis, electron energy filtered imaging analysis, and electron microscope autoradiography. The data were demonstrated as elemental distribution images and were calculated quantitatively. For the soluble low molecular peptides, we developed a tannic acid and aldehyde method combined with microwaved fixation. We discuss the theoretical background of the tannic acid fixation and microwaved fixation methods. For the organic solvent soluble substances, i.e., lipids including steroids, we successfully tested the use of a mixed fixative of aldehyde and osmium, digitonization, and osmification with the use of p-phenylendiamine or imidazole. We also proposed some new ideal biotracers for electron beam X-ray microanalysis and electron energy filtered imaging analysis.
我们测试了各种固定和分析方法，以通过电子显微镜对组织和细胞中的元素成像进行展示，即多种离子元素、水溶性低分子肽等可溶性物质，甚至脂质等有机溶剂可溶性物质。对于离子元素，我们测试了冷冻干燥或冷冻替代方法以及与微波固定方法相结合的有机或无机特殊化学沉淀方法。数据通过电子束X射线微分析、电子能量过滤成像分析和电子显微镜放射自显影进行分析。数据以元素分布图像的形式展示并进行定量计算。对于可溶性低分子肽，我们开发了一种结合微波固定的单宁酸和醛法。我们讨论了单宁酸固定和微波固定方法的理论背景。对于有机溶剂可溶性物质，即包括类固醇在内的脂质，我们成功测试了使用醛和锇的混合固定剂、洋地黄皂苷处理以及使用对苯二胺或咪唑进行锇化处理。我们还提出了一些用于电子束X射线微分析和电子能量过滤成像分析的新型理想生物示踪剂。
