Electron Tomography: A 3D View of the Subcellular World
Kenneth H. Downing Haixin Sui Manfred Auer Lawrence Berkeley National Laboratory
A convergence of technological advances has enabled biological applications of electron tomography, thereby expanding the range of accessible size scales and complexity.
© 2007 American Chemical Societ y
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lectron microscopy (EM) revolutionized cell biology in the 1960s when it revealed details of cellular ultrastructure. Sections of cells were preserved well enough and cut thin enough to allow examination with the electron microscope at a resolution ~100× better than was possible by light microscopy. With improvements in light microscopes and the advent of genetically encoded fluorescent proteins during the past 20 years, however, EM has taken a backseat as fluorescence microscopy became the dominant method for studying cellular processes; this was due in part to light microscopy’s ability to image the dynamic behavior of proteins in live cells. N o v e m b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y
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EM is now making a significant resurgence as electron tomography opens a wide range of new explorations of cellular ultrastructure in 3D at molecular resolution. Tomography is providing information not only on the location and interactions of subcellular components but also on the structure of these molecular components within the cell and in isolated complexes. Several recent reviews have discussed the technologies involved and the broad range of applications (1, 2). We have come a long way since the early days of ultrastructure description and learned to appreciate that cellular activities rely on the functional integration of macromolecular, multiprotein complexes and their precise localization within the cell. For example, metabolic and signaling pathways are often confined to certain subcellular sites, and the assembly of the components into multiprotein complexes results in a higher efficiency than if cellular chemistry were left to random encounters of substrates and enzymes. These systems are often viewed as molecular “machines” composed of a number of parts, each with a distinctive function (3). The subunit composition, distribution, and potential interactions of these macromolecular complexes, as well as of the cytoskeletal structures that regulate their locations, are prime targets of current electron tomographic studies. Although resolution in light microscopy has improved significantly (4, 5), commonly used confocal imaging still has a resolution limit of ~250–400 nm, which does not provide much information about the cellular context. At the other end of the resolution spectrum, X-ray crystallography, NMR spectroscopy, and cryo-EM of isolated particles and 2D crystals have produced a large number of protein structures at or near atomic resolution. Electron tomography has the potential to fill the gap between global cellular localization and detailed 3D molecular structure, because it can reveal the localization within the cellular context at true molecular resolution and the shapes and 3D architecture of large molecular machines. It can reveal also the interaction of individual proteins and protein complexes with other cellular components, such as DNA and membranes. The combination of high-resolution methods (which excel in determining the atomic structure), electron tomography (which allows determination of the precise subcellular positions and interactions of macromolecular machines), and fluorescence-based light microscopy (which describes the dynamic nature of cellular processes) promises to lead to an integrated and profound understanding of cellular processes for the physiological function of tissues and organs and for the pathogenesis of disease.
Electron tomography
A tomogram is a 3D volume computed from a series of projection images that are recorded as the object in question, whether a human body or a molecule, is tilted at different orientations. Some of the technological developments that have 7950
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enabled the application of tomography to EM are related to the advances that have made computed axial tomography (CAT) scans a routine diagnostic tool—faster computers with greater memory and efficient and accurate methods for computing the volume and visualizing the result. Automation of EM instrumentation has allowed minimal operator oversight for managing the long and tiresome procedure of recording typically 50–150 images in the tilt series (6). Intermediate-voltage microscopes, operating at 200–400 kV, provide the ability to examine specimens that are thicker and more rewarding to examine in three dimensions. The development of efficient CCD cameras for EM has eliminated the tedium and delay of processing and digitizing images recorded on photographic film. Because of the relative ease with which electron tomography can now be implemented, explosive growth has occurred during the past few years in the number of groups using tomography and in the range of its applications. In the biological sciences, applications are divided into two classes that use somewhat different techniques for preparing specimens. The first approach involves sectioning materials that are too thick for the electron beam to penetrate completely. Samples made by plastic embedding, sectioning, and staining have been used widely to study the morphology, ultrastructure, and content of cells and their subcellular organelles (2, 7 ). The development of ultrarapid freezing brought a tremendous improvement in preservation, and this is one reason that these structures can be resolved in three dimensions (8). Frozen specimens often are freeze-substituted, resin-embedded, and contrasted with heavy metals, which deposit around cellular components. Although this process may limit the resolution and prevent detection of secondary-structure elements within the proteins, it is sufficient to provide the shapes of large molecular complexes (9). As a variant of this approach, the frozen sample also can be cut thinly into vitreous sections for cryo-EM studies without plastic embedding or staining. Vitreous sections have inherently low contrast, are difficult to orient precisely, and may crack because of compression, which makes this approach less attractive for some biological problems (10). However, some results from cryo-sectioned bacteria samples have been spectacular, particularly where the preservation of membrane structure and extracellular glycoprotein architecture are concerned (11). The second approach avoids sectioning and staining altogether. If specimens are thin enough that sectioning is not required—for example, isolated protein complexes, thin bacteria, or some extended processes of larger cells—imaging in the frozen-hydrated and unstained state offers an artifact-free view of the structure. The preparation of frozen-hydrated specimens by rapid plunging into liquid ethane preserves the native, hydrated structure by vitrifying the water. This procedure has become routine in EM studies of individual proteins and has been adapted easily to tomography. Electron tomography is suited ideally to imaging isolated particles that are too complex and heterogeneous to be studied by cryo-EM single-particle analysis. With either sample preparation approach, the two major
goals are to determine the detailed shape and structure of organelles and molecular complexes and to ascertain the location of particular components within the cellular context. Often these goals overlap substantially. The forte of tomography is the study of systems that are unique, such as molecular complexes that are found either in a variety of conformations or in such a large context (i.e., the cell) that no one environment is exactly like any other. These instances are often like snapshots representing the operational stages in the working of the machines. For large complexes that cannot be crystallized for high-resolution study, tomography allows determination of the structure at a moderate resolution so that various known components can be docked together to reveal how the complex is built and how the pieces move. From an inventory of the parts, we can learn how they relate to each other and interact. Tomograms often present views of many complexes in the same state, especially for purified samples. Averaging many images of identical objects, as is done in cryoEM studies of protein structure, can greatly improve S/N and the visibility of features in the structure. Hydrated, unstained proteins are highly sensitive to damage by the electron beam, so the exposure of the specimen is low and results in images that are statistically poorly defined. Stained specimens generally show a granular or irregular distribution of the heavy metal. In both cases, averaging many equivalent volumes extracted from the 3D reconstructions improves the statistics and S/N, thus yielding improved resolution.
The procedures
For electron tomography to be used, the biological question needs to be formulated so that the technique is applicable. In addition, faithful sample preservation is needed. Specimens 1 mm thick are often subject to severe fixation artifacts likely due to metabolic starvation; this results in visible alterations of membrane architecture. Although the instruments possess advanced automation software, the operator must be competent with the instrument; 50–150 images are recorded as the sample is tilted through as large a range as possible, often ±70°. For frozen-hydrated samples, the total exposure needs to be
