Conventional transmission electron microscopy

摘要

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project. Transmission electron microscopy (TEM) has been an important technology in cell biology ever since it was first used in the early 1940s. The most frequently used TEM application in cell biology entails imaging stained thin sections of plastic-embedded cells by passage of an electron beam through the sample such that the beam will be absorbed and scattered, producing contrast and an image (see Table 1 for a definition of terms). Because of the short wavelength of the electron beam (100,000-fold shorter than photons in visible light), TEM can achieve subnanometer resolution—well below that of even the highest-resolution light microscopes, ～20 nm. Similar to our longer-wavelength light-microscopy brethren, electron microscopists are faced with a growing array of instruments for specimen preparation and imaging. The emerging technologies focus on preserving the native ultrastructure of samples by vitrification, achievement of higher resolution, three-dimensional ultrastructure by electron tomography, and precise correlation of specific cell structures using immunofluorescence microscopy and TEM (correlative light and electron microscopy [CLEM]). The combination of these techniques is being used to cover the complete spectrum of structure, from molecular to whole cell. Here we discuss conventional TEM of thin sections for morphology and immunolocalization. Even when the EM is done with the assistance of a core facility, we suggest that it is worthwhile for cell biologists who propose to use TEM in their studies to be well enough versed in the specimen preparation and imaging technologies to design appropriate experiments and be able to participate in trouble shooting when necessary. TABLE 1: Talk EM Like a Pro. TEM has proven valuable in the analysis of nearly every cellular component, including the cytoskeleton, membrane systems, organelles, and cilia, as well as specialized structures in differentiated cells, such as microvilli and the synaptonemal complex. There is simply no way to visualize the complexity of cells and see cellular structures without TEM. Despite its power, the use of TEM does have limitations. Among the limitations are the relatively small data set of cells that can be imaged in detail, the obligate use of fixed—therefore deceased—cells, and the ever-present potential for fixation and staining artifacts. However, many of these artifacts are well known and have been catalogued (e.g., Bozzola and Russell, 1999 ; Maunsbach and Afzelius, 1999) . A typical TEM experiment consists of two phases: the live-cell experiment, in which a cell type, possibly a mutant, is grown under given conditions for analysis, followed by preparation of the specimen and imaging by TEM. Specimen preparation for conventional TEM is comprehensively reviewed in Hayat (1970) and briefly described here ( Figure 1 ). FIGURE 1: A brief flowchart showing the work to be done with different types of sample preparation for conventional electron microscopy (yellow background). The advanced cryo-EM techniques are shown with a blue background. For immuno-EM, the samples can be stained ...