Lea P J, Hollenberg M J, Temkin R J, Khan P A
Department of Anatomy, Faculty of Medicine, University of Toronto, Ontario, Canada.
Microsc Res Tech. 1992 Jul 1;22(2):185-93. doi: 10.1002/jemt.1070220207.
Detailed examination of subcellular structures in three dimensions (3D) by high resolution scanning electron microscopy (HRSEM) is now possible due to improvements in the design of the scanning electron microscope and the introduction of methods of specimen preparation using chemical removal of the cytosol and cytoskeleton by dilute osmium tetroxide. Cells which have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction display intact intracellular structures in 3D including nuclear chromatin, endoplasmic reticulum, mitochondria, and the Golgi complex at a resolution close to that of conventional biological transmission electron microscopy (TEM). Small changes in the 3D structure of subcellular components can be conveniently examined in this way in development, in a variety of physiological processes and in disease. Broad areas of the specimen can be quickly surveyed by HRSEM since sectioning is not required and specimens of comparatively large size (up to 5 mm3) can be placed in the microscope. Extraction of the cytosol with dilute osmium tetroxide (OsO4) exposes subcellular structures in relief, permitting their examination in 3D from several aspects. However, the OsO4 extraction technique is limited, since significant intracellular structures, such as the cytoskeleton, vesicles, and antibody binding sites can be removed or inactivated during the cytosol removal steps.
由于扫描电子显微镜设计的改进以及采用稀四氧化锇化学去除细胞质和细胞骨架的标本制备方法的引入，现在可以通过高分辨率扫描电子显微镜（HRSEM）对亚细胞结构进行三维（3D）详细检查。经过固定、冷冻、切割、解冻并进行细胞质提取的细胞，在三维空间中呈现出完整的细胞内结构，包括核染色质、内质网、线粒体和高尔基体复合体，其分辨率接近传统生物透射电子显微镜（TEM）。通过这种方式，可以方便地在发育过程、各种生理过程和疾病中检查亚细胞成分三维结构的微小变化。由于不需要切片，并且可以将相对较大尺寸（高达5立方毫米）的标本放入显微镜中，HRSEM可以快速扫描标本的广阔区域。用稀四氧化锇（OsO4）提取细胞质可使亚细胞结构凸显出来，从而可以从多个方面对其进行三维检查。然而，OsO4提取技术存在局限性，因为在去除细胞质的步骤中，重要的细胞内结构，如细胞骨架、囊泡和抗体结合位点可能会被去除或失活。
