Miyai K, Abraham J L, Linthicum D S, Wagner R M
Lab Invest. 1976 Oct;35(4):369-76.
Several methods of tissue preparation and different modes of operation of the scanning electron microscope were used to study the ultrastructure of rat liver. Rat livers were perfusion fixed with buffered 2 per cent paraformaldehyde or a mixture of 1.5 per cent paraformaldehyde and 1 per cent glutaraldehyde and processed as follows. Tissue blocks were postfixed in buffered 2 per cent osmium tetroxide followed sequentially by the ligand-mediated osmium binding technique, dehydration and cryofracture in ethanol, and critical point drying. They were then examined without metal coating in the scanning electron microscope operating in the secondary electron and backscattered electron modes. Fifty-micrometer sections were cut with a tissue sectioner, stained with lead citrate, postfixed with osmium, dehydrated, critical point dried, and examined in the secondary electron and back-scattered electron modes. Frozen sections (0.25 to 0.75 mum. thick) were cut by the method of Tokuyasu (Toluyasu KT: J Cell Biol 57:551, 1973) and their scanning transmission electron microscope images were examined either with a scanning transmission electron microscope detector or with a conversion stub using the secondary electron detector. Secondary electron images of the liver prepared by ligand-mediated osmium binding and subsequent cryofracture revealed such intracellular structures as cisternae of the endoplasmic reticulum, lysosomes, mitochondria, lipid droplets, nucleolus and nuclear chromatin, as well as the usual surface morphology, Lipocytes in the perisinusoidal space were readily identified. Backscattered electron images. Unembedded frozen sections had little drying artifact and were virtually free of freezing damage. The scanning transmission electron microscope image revealed those organelles visualized by the secondary electron mode in the ligand-mediated osmium binding-treated tissue.
采用几种组织制备方法和扫描电子显微镜的不同操作模式来研究大鼠肝脏的超微结构。大鼠肝脏用2%多聚甲醛缓冲液或1.5%多聚甲醛与1%戊二醛的混合物进行灌注固定，并按以下方法处理。组织块先用2%四氧化锇缓冲液后固定，随后依次采用配体介导的锇结合技术、在乙醇中脱水和冷冻断裂，以及临界点干燥。然后在以二次电子和背散射电子模式运行的扫描电子显微镜中不进行金属镀膜检查。用组织切片机切取50微米厚的切片，用柠檬酸铅染色，用锇后固定，脱水，临界点干燥，并在二次电子和背散射电子模式下检查。采用德久安寿（Tokuyasu KT: J Cell Biol 57:551, 1973）的方法切取冷冻切片（0.25至0.75微米厚），并用扫描透射电子显微镜探测器或使用二次电子探测器的转换短柱检查其扫描透射电子显微镜图像。通过配体介导的锇结合和随后的冷冻断裂制备的肝脏二次电子图像显示出细胞内结构，如内质网池、溶酶体、线粒体、脂滴、核仁和核染色质，以及通常的表面形态，窦周间隙中的脂肪细胞很容易识别。背散射电子图像。未包埋的冷冻切片几乎没有干燥伪像，并且几乎没有冷冻损伤。扫描透射电子显微镜图像显示了在配体介导的锇结合处理组织中通过二次电子模式可视化的那些细胞器。
