Scala C, Cenacchi G, Preda P, Vici M, Apkarian R P, Pasquinelli G
Institute of Clinical Electron Microscopy, S. Orsola Hospital, University of Bologna, Italy.
Scanning Microsc. 1991 Mar;5(1):135-44; discussion 144-5.
Intracellular structures of embedded biological tissues (rat kidney, myocardium and small intestine) were observed by conventional-scanning electron microscopy (C-SEM) and high-resolution scanning electron microscopy (HR-SEM) after glass knife sectioning. C-SEM of semi-thin sections of material processed the same way as conventional transmission electron microscopy (TEM) provided strong backscattered electron (BSE)-dependent, two-dimensional secondary electron images (SEI(-)) which precisely integrated and further extended previous light microscopy (LM) observation of the same specimen. In addition, the three-dimensional (3-D) arrangement of intracellular organelles was appreciated using a mixture of acetone-soluble acrylic resin in place of epoxy resin embedding. Since the identification of such structures was hampered by the use of conventional fixations we introduced osmium maceration as a preliminary step to remove excess cytoplasmic matrix from the specimen. Consequently, semi-thin sections for LM and thin sections for TEM were obtained by sectioning of the tissue blocks. After resin removal, the sections were successfully observed in 3-D under a C-SEM. Finally, the deembedded, osmium treated sections proved to be smooth enough to facilitate deposition of continuous, ultra-thin (1 nm) chromium films and, therefore, HR-SEM studies of macromolecular cell membrane structures.
在使用玻璃刀切片后，通过常规扫描电子显微镜（C-SEM）和高分辨率扫描电子显微镜（HR-SEM）观察嵌入生物组织（大鼠肾脏、心肌和小肠）的细胞内结构。以与传统透射电子显微镜（TEM）相同的方式处理材料的半薄切片的C-SEM提供了强烈的背散射电子（BSE）依赖的二维二次电子图像（SEI(-)），该图像精确整合并进一步扩展了先前对同一标本的光学显微镜（LM）观察。此外，使用丙酮可溶性丙烯酸树脂代替环氧树脂包埋来观察细胞内细胞器的三维（3-D）排列。由于使用传统固定方法会妨碍对这些结构的识别，我们引入锇浸渍作为初步步骤，以从标本中去除多余的细胞质基质。因此，通过对组织块进行切片获得用于LM的半薄切片和用于TEM的薄切片。去除树脂后，这些切片在C-SEM下成功进行了三维观察。最后，经脱包埋、锇处理的切片足够光滑，便于沉积连续的超薄（1 nm）铬膜，因此可用于对大分子细胞膜结构进行HR-SEM研究。
