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Establishment of embedment-free electron microscopy and analysis of the nature of cytoplasmic matrix by this methodology

免包埋电子显微镜的建立及利用该方法分析细胞质基质的性质

基本信息

批准号：
62480092
负责人：
KONDO Hisatake
金额：
$ 4.29万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Using polyethylene glycol (PEG), a highly water soluble wax as a transient embedding media, embedment-free sections of chemically fixed tissues are obtained reliably for the electron microscopic examination. After critical-point drying with CO_2, these embedment-free sections present the same aspect of cell ultrastructure as whole mounted, fixed, and dried cultured cells, and filamentous elements (microtrabeculae), which have been unrecognized before in the conventional cytoplasmic matrix, are clearly revealed. Wehen the embedment-free sections are rotaly-replicated with platinum and carbon, resulting images of the sections are of high quality, with excellent resolution and to be quite comparable to those obtained with the rapid freezing, deep etched replica method.With this new methodology, the membrane specializations on the outer and inner cell surfaces as well as the organization of the cytoskeleton is clearly demonstrated. However, after the attainment of the intracellular displacement of cell organelles and some proteins by ultracentrifugation, no marked change in organization of the microtrabecular lattice is discerned.This together with my previous finding that artificial protein solutions at certain concentration exhibit meshworks quite similar to the intracellular microtrabeculae,argue against the idea that gthe filamentous or midrotrabecular strands are the real structure of living cells. Regerdless of the reality or artifact of the microtrabeculae, the PEG method is also applicable to general scanning electron microsoopy, and the intermicroscopic correlation of images between scanning and transmission electron microscopy and light microscopy is easily and reliably performed. Furthermore, the PEG-method is shown to be suitable for light and electron microscopic immunocytochemistry. Therefore this methodology should prove to be valuable adjunct to conventional microscopic techniques.

使用聚乙二醇（PEG）（一种高度水溶性的蜡）作为瞬时包埋介质，可以可靠地获得化学固定组织的无包埋切片，用于电子显微镜检查。经过CO_2临界点干燥后，这些免包埋切片呈现出与完整安装、固定和干燥的培养细胞相同的细胞超微结构，并且清晰地揭示了以前在常规细胞质基质中无法识别的丝状成分（微小梁）。当使用铂和碳旋转复制免包埋切片时，所得到的切片图像质量高、分辨率高，与快速冷冻、深蚀刻复制方法获得的图像相当。通过这种新方法，可以清楚地展示细胞外表面和内表面的膜特化以及细胞骨架的组织。然而，通过超速离心实现细胞器和一些蛋白质的细胞内置换后，微小梁晶格的组织没有明显变化。这与我之前发现的一定浓度的人工蛋白质溶液表现出与细胞内微小梁非常相似的网状结构相结合，反对丝状或中小梁的观点链是活细胞的真实结构。无论微小梁的真实性或伪影如何，PEG方法也适用于一般扫描电子显微镜，并且扫描和透射电子显微镜与光学显微镜之间的图像的显微相关性可以容易且可靠地进行。此外，PEG 方法被证明适用于光和电子显微镜免疫细胞化学。因此，这种方法应该被证明是传统显微技术的有价值的辅助手段。