Hendricks Gregory M
Core Electron Microscopy Facility, Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA.
Methods Mol Biol. 2014;1117:73-93. doi: 10.1007/978-1-62703-776-1_5.
Metal shadowing of bacteria, viruses, isolated molecules, and macromolecular assemblies is another high-resolution method for observing the ultrastructure of biological specimens. The actual procedure for producing a metal shadow is relatively simple; a heavy metal is evaporated from a source at an oblique angle to the specimen. The metal atoms pile up on the surfaces that face the source, but the surfaces away from the source are shielded and receive little metal deposit, creating a "shadow." However, the process of producing biological specimens that are suitable for metal shadowing can be very complex. There are a whole host of specimen preparation techniques that can precede metal shadowing, and all provide superior preservation in comparison to air drying, a required step in negative staining procedures. The physical forces present during air drying (i.e., surface tension of the water-air interface) will literally crush most biological specimens as they dry. In this chapter I explain the development of and procedures for the production of biological specimens from macromolecular assemblies (e.g., DNA and RNA), purified isolated molecules (e.g., proteins), and isolated viruses and bacteria preparations suitable for metal shadowing. A variation on this basic technique is to rotate the specimen during the metal deposition to produce a high-resolution three-dimensional rendering of the specimen.
对细菌、病毒、分离出的分子以及大分子聚集体进行金属投影是观察生物标本超微结构的另一种高分辨率方法。产生金属投影的实际过程相对简单；重金属从一个源以倾斜角度蒸发到标本上。金属原子堆积在面对源的表面上，但远离源的表面被屏蔽，几乎没有金属沉积，从而形成一个“投影”。然而，制备适合金属投影的生物标本的过程可能非常复杂。在金属投影之前有一整套标本制备技术，并且与负染色程序中必需的空气干燥步骤相比，所有这些技术都能提供更好的保存效果。空气干燥过程中存在的物理力（即水 - 空气界面的表面张力）在标本干燥时实际上会压碎大多数生物标本。在本章中，我将解释从大分子聚集体（如DNA和RNA）、纯化的分离分子（如蛋白质）以及适合金属投影的分离病毒和细菌制剂制备生物标本的发展过程和程序。这种基本技术的一个变体是在金属沉积过程中旋转标本，以生成标本的高分辨率三维图像。
