Ruiz Teresa, Radermacher Michael
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, USA.
Methods Mol Biol. 2006;319:403-25. doi: 10.1007/978-1-59259-993-6_19.
Electron microscopy of single particles has recently become a very popular field in both biological and material sciences. It might be difficult for a novice researcher new to this field to know how to start tackling a new project. This chapter is designed to serve as a guideline for anyone starting a new project to determine a three-dimensional structure using single-particle techniques. The chapter describes the basic techniques necessary to prepare the samples and acquire the data to calculate a three-dimensional reconstruction in easy-to-understand, step-by-step instructions. It starts with the basic preparation of support films and the usage of a variety of staining techniques needed to assess the quality of the sample and the viability of the project. It ends with a detailed description of vitreous ice preparations designed to acquire high-resolution structural information. Guidelines and tips are given on how to record the best images with an electron microscope. Although this chapter is geared to researchers new to the field, experts might find it not only useful as a reference but also valuable because of the number of practical tips included.
单颗粒电子显微镜技术最近在生物科学和材料科学领域都成为了一个非常热门的领域。对于刚涉足该领域的新手研究人员来说，可能很难知道如何着手开展一个新项目。本章旨在为任何使用单颗粒技术确定三维结构的新项目启动者提供指导方针。本章以易于理解的分步说明描述了制备样品和获取数据以计算三维重建所需的基本技术。它从支撑膜的基本制备以及评估样品质量和项目可行性所需的各种染色技术的使用开始。最后详细描述了旨在获取高分辨率结构信息的玻璃态冰制备方法。文中还给出了关于如何用电子显微镜记录最佳图像的指导方针和提示。尽管本章是为该领域的新手研究人员编写的，但专家们可能会发现它不仅作为参考很有用，而且由于其中包含的实用提示数量众多，也很有价值。
