Suzuki E
Division of Structural Biology, Department of Basic Medical Science, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
J Microsc. 2002 Dec;208(Pt 3):153-7. doi: 10.1046/j.1365-2818.2002.01082.x.
The feasibility of plasma coating of a thin osmium layer for high-resolution immuno-scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro-muscular preparations were fixed with formaldehyde and labelled with a neurone-specific antibody and 10 or 5 nm colloidal gold-conjugated secondary antibodies. The specimens were post-fixed with osmium tetroxide and freeze-dried. Then they were coated with a 1-2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three-dimensionally complex surface structures of cellular processes with a resolution of several nanometres.
以果蝇胚胎运动神经元作为模型系统，测试了用于细胞表面高分辨率免疫扫描电子显微镜的薄锇层等离子体涂层的可行性。神经肌肉标本用甲醛固定，并用神经元特异性抗体和10或5纳米胶体金偶联的二抗标记。标本用四氧化锇后固定并冷冻干燥。然后使用空心阴极等离子体镀膜机用1-2纳米厚的锇层进行镀膜。非晶态锇的薄而连续的涂层在背散射电子图像中同时给出了金颗粒的良好信号和神经突的精细表面结构。这种方法使得以几纳米的分辨率可视化抗原分布和细胞突起的三维复杂表面结构成为可能。
