Haggis G H
Research Branch, Agriculture Canada, Ottawa.
Microsc Res Tech. 1992 Jul 1;22(2):151-9. doi: 10.1002/jemt.1070220204.
Methods are reviewed for examination of internal cell structure by high-resolution scanning electron microscopy and compared with the rapid-freeze deep-etch replica technique used in transmission electron microscopy. Rapid freezing of fresh material, followed by freeze-fracture, provides a theoretically attractive approach in ultrastructure studies, but the high protein and solute content of most cells prevents a deep three-dimensional view for material frozen without some form of extraction. After discussion of other methods it is concluded that the most useful general approach, at least for cultured cells, is to first permeabilize or break open the cells in a medium which preserves the structure under study in a functional state as, for example, the movement of chromosomes along the division spindle, or transport of proteins within the Golgi region. After permeabilization, with attendant partial extraction, the preparation can be fixed, then viewed by either deep-etch replication, or by high-resolution scanning electron microscopy, with structure of interest revealed in deep view.
本文回顾了通过高分辨率扫描电子显微镜检查细胞内部结构的方法，并与透射电子显微镜中使用的快速冷冻深蚀刻复型技术进行了比较。新鲜材料的快速冷冻，随后进行冷冻断裂，在超微结构研究中提供了一种理论上有吸引力的方法，但大多数细胞的高蛋白和溶质含量使得在没有某种形式提取的情况下冷冻的材料无法获得深度三维视图。在讨论了其他方法后得出结论，至少对于培养细胞来说，最有用的一般方法是首先在一种能将所研究结构保持在功能状态的培养基中使细胞通透或破裂，例如染色体沿分裂纺锤体的移动，或蛋白质在高尔基体区域内的运输。通透后，伴随部分提取，制备物可以固定，然后通过深蚀刻复型或高分辨率扫描电子显微镜观察，感兴趣的结构以深度视图显示。
