Cryoelectron Tomography (CET)
More than 70 years have now passed since the invention of the first transmission electron microscope (Knoll and Ruska, 1932; Ruska, 1987). Many reports and publications have been written covering the technological achievements in electron microscopy (EM) and more important the scientific breakthroughs related to the information disclosed by EM studies. EM, in its various “flavors” (see below), is now a wellestablished method in life as well as in material science. Therefore, almost every laboratory in the field of structural analysis is equipped with one or several low or intermediate voltage microscopes for routine use and in some instances for high-end applications. However, compared to light microscopy, EM is still a “young” method but with great potential for further improvements. This has been shown, especially in the past decade: among the highlights are the incorporation of energy filters, monochromators, aberration correctors, and above all the almost complete replacement of negative plates by charge coupled device (CCD) cameras and imaging plates. By all means, these technical achievements have been supported by the development of the computer and its availability throughout science. The various techniques in EM, such as bright field (BF) or dark field (DF) imaging, weak beam imaging, conventional transmission EM (CTEM), high-resolution TEM or EM (HRTEM or HREM), scanning transmission electron microscopy (STEM), energy-filtered TEM (EFTEM), and many others, have been improved and, moreover, expanded with the computer power on hand. Today’s most modern techniques are all based on automated ac quisition and alignment procedures, reconstruction algorithms, and especially on elaborate image processing routines. Here, we address the advances made in biological EM and especially in cryo-EM. Biological structures can be, by and large, characterized as pleiomorphic. Just as every human being has a different face, cells, proteins, and macromolecular complexes have different shapes and forms, designed for a higher functional purpose. It is far from random and instead of addressing them as “amorphic” or “amorphous,” as in physics for randomly ordered solids, they are called “pleiomorphic” or “pleiomorph.” Moreover, the inside of a cellular structure resembles the image of a giant factory, where the single constituents act together, building highly specific molecular machines and, if necessary, change their purpose (Alberts, 1998). Therefore it is a highly variable and dynamic environment. Every intrusion into this fragile system can lead to changes. The suitable preparation for a final characterization with the EM is a major challenge. And it is likewise a challenge in terms of the environment inside EMs: an ultrahigh vacuum and electron radiation.
KeywordsTilt Angle Complementary Metal Oxide Semiconductor Fourier Space Charge Couple Device Camera Algebraic Reconstruction Technique
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