Transmission Electron Microscopy of Biological Samples

Abstract

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 highresolution 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 cryotransmission electron microscopy (cryo-TEM), while thicker samples require a thinning step via cryo-electron microscopy of vitreous sections (CEMOVIS) or cryofocused 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 anoxiasensitive 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.

Mitochondrial Morphology and Mitophagy in Heart Diseases: Qualitative and Quantitative Analyses Using Transmission Electron Microscopy

“…Since the development of the first electron microscope in the 1930's, transmission electron microscopy (TEM) has been an essential technique for the visualization of cellular ultrastructure. The timeline of the key events in the development and evolution of TEM has been described in an previous study (Lukasz Mielanczyk et al, 2015). TEM has steadily gained popularity in the cardiovascular research field over the last 20 years, being utilized in over 2,000 peer-reviewed cardiovascular research publications (Figure 1), to examine cardiac ultracellular organization at the nanometer scale.…”

“…In this review, we discuss the application of TEM as a tool for viewing cardiac ultrastructure and how this has been leveraged in cardiovascular studies to visualize complex signaling processes, with a special focus on the mitochondria. Although it is important to appreciate that, in addition to TEM, scanning electron microscopy (SEM) has also been used in several cardiovascular studies, its primary application is to provide information on cell surface and composition, rather than cellular ultrastructure; therefore, SEM will not be discussed in this review (Klang et al, 2012;Lukasz Mielanczyk et al, 2015).…”

Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

“…Alternatively, cryofixation or high-pressure freezing is used. These techniques solidify the specimens so that they can be cut to the desired thickness but at the same time hamper antibody penetration and, depending on the protocol, alter epitopes of proteins of interest by denaturation [18]. Moreover, the setup and its maintenance are expensive and experienced personnel is required to reach an acceptable throughput [19].…”

STEM-EELS investigation of c-Si/a-AlO interface for solar cell applications

“…These characterizations could be improved using TEM techniques that are applied in biology filed such as e.g. : cryo-TEM, cryo-sample-holder or low dose camera [65].…”