Chapters authored
Transmission Electron Microscopy of Biological Samples
During the last 70 years, transmission electron microscopy (TEM) has developed our knowledge about ultrastructure of the cells and tissues. Another aim is the determination of molecular structure, interactions and processes including structure-function relationships at cellular level using a variety of TEM techniques with resolution in atomic to nanometre range. Even with the best transmission electron microscope, it is impossible to obtain real results without optimal sample preparation, respecting both the structure and the antigenicity preservation. Preparation techniques for high-resolution study of both macromolecular complex and organelles within cellular complex are based on fast cryoimmobilisation process, where the sample is in the most native, hydrated state. Next, thin samples are directly visualised under cryo-transmission electron microscopy (cryo-TEM), while thicker samples require a thinning step via cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-focused ion beam (cryo-FIB) before visualisation. Alternatively, vitrified samples are freeze substituted and embedded in chosen resin for room temperature ultramicrotomy. This preparation technique is suitable for morphological study, 3D analysis of cellular interior and immunoelectron microscopy. A different route for immunolocalisation study is cryosectioning according to the Tokuyasu technique that is a choice for rare or methacrylate-sensitive antigens. Most recently, new hybrid techniques have been developed for difficult-to-fix organisms and antigens or labile and anoxia-sensitive tissues. Another preparation technique is, the oldest but still important, conventional chemical fixation dedicated in a wide range of research interest, involving morphological and immunolocalisation study. In this chapter, we present different sample preparation approaches for transmission electron microscopy of biological samples, including its methodological basis and applications.
Part of the book: The Transmission Electron Microscope