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Correspondence to: Bruce F. McEwen, Wadsworth Center, New York State Dept. of Health, PO Box 509, Albany, NY 12201-0509. E-mail: bruce.mcewen@wadsworth.org
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Summary |
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Electron tomography has emerged as the leading method for the study of three-dimensional (3D) ultrastructure in the 520-nm resolution range. It is ideally suited for studying cell organelles, subcellular assemblies and, in some cases, whole cells. Tomography occupies a place in 3D biological electron microscopy between the work now being done at near-atomic resolution on isolated macromolecules or 2D protein arrays and traditional serial-section reconstructions of whole cells and tissue specimens. Tomography complements serial-section reconstruction by providing higher resolution in the depth dimension, whereas serial-section reconstruction is better able to trace continuity over long distances throughout the depth of a cell. The two techniques can be combined with good results for favorable specimens. Tomography also complements 3D macromolecular studies by offering sufficient resolution to locate the macromolecular complexes in their cellular context. The technology has matured to the point at which application of electron tomography to specimens in plastic sections is routine, and new developments to overcome limitations due to beam exposure and specimen geometry promise to further improve its capabilities. In this review we give a brief description of the methodology and a summary of the new insights gained in a few representative applications.
(J Histochem Cytochem 49:553563, 2001)
Key Words: electron tomography, transmission electron, microscopy, three-dimensional, reconstruction, plastic embedding, cellular ultrastructure
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Introduction |
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TRANSMISSION ELECTRON MICROSCOPY (TEM) has been largely responsible for shaping the modern view of cellular architecture. Many of the seminal studies were conducted during the late 1950s and the 1960s in conjunction with the development of plastic embedding, ultramicrotomy, and appropriate fixatives and stains. Although it remained an important diagnostic tool, during the 1970s and 1980s TEM's stature in basic research was largely overshadowed by rapid advancements in biochemistry, molecular biology, genetics, and light microscopy. Nevertheless, significant technical developments continued during that period, with the result that TEM re-emerged in the 1990s in a spectacular way as a crucial tool for understanding molecular mechanisms in cellular biology.
Microscope improvements include computer control, improved specimen stages, high-sensitivity digital image recording, field-emission electron sources, and energy filtering. Combined with increased computational power and developments in image-processing software, two important applications of biological TEM for 3D structure determination have emerged. The first, and currently the major application, is high-resolution biological electron microscopy (reviewed by
In the high-resolution applications, (e.g., 3D resolution better than 1 nm), TEM complements X-ray crystallography and nuclear magnetic resonance (NMR) studies. This was dramatically illustrated by the recent use of electron crystallography to determine the atomic structure of the tubulin dimer, a key cytoskeletal component involved in many cell processes (
As impressive as this docking approach has been, its application has thus far been limited to a small subset of cell structures. The reason for this is that high- and medium-resolution structural determination, whether by X-ray crystallography, NMR, or TEM, requires averaging thousands of copies of an "identical" motif (e.g., protein molecules in a 3D or 2D crystal lattice, subunits of a helix, or computationally aligned particles such as ribosomes). Because most organelles and cellular substructures are complex pleomorphic assemblies that show considerable individual variation, they cannot be averaged as identical motifs. Electron tomography is the only computational 3D reconstruction technique not based on averaging. Therefore, it is generally the method of choice for 3D reconstruction of subcellular structures. Although resolution is necessarily limited by the lack of averaging, 3D resolution of 510 nm is currently obtained. This enables tomography to bridge the gap between serial section reconstruction and the higher-resolution approaches (see below). The ultimate goal of electron tomography is to compute 3D structural maps of cell organelles with sufficient resolution to locate "molecular signatures" of individual macromolecules and thereby enable the docking approach to be extended to a much wider range of macromolecules and cell substructures.
Here we provide a general introduction to electron tomography, along with a summary of selected applications during the previous decade and a brief look into current developments.
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General Approach of Electron Tomography |
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Background for 3D Imaging
Like all forms of imaging that use penetrating radiation, TEM provides a translucent or "X-ray" view of the specimen. Although this has the advantage of enabling the user to see inside the structure, it also has the disadvantage that structural details from different depths in the specimen are superimposed in a 2D projection. This process of image formation is illustrated in Fig 1A, in which a 2D "smiley face" is projected onto a 1D line. Peaks of mass are recorded in those segments of the line where the imaging rays transverse through an eye or the nose in addition to the mouth. Such images are confusing and counterintuitive, both because details are superimposed upon one another and because we are accustomed to viewing surfaces in the everyday world. That is why the surface views provided by scanning electron microscope images are easier to interpret than TEM images.
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The traditional approach to circumvent the problem of superimposed features in the 2D images is to embed specimens in plastic and cut sections so thin (i.e., 4080 nm thick) that each section is effectively a 2D slice from the specimen. When the pitfalls of structural interpretation from 2D information became apparent, methods were developed for 3D reconstruction via stacking serial thin sections (e.g.,
Electron tomography uses the opposite approach to solve the 2D projection problem. Plastic sections are cut thick enough (2001000 nm) to contain a significant amount of information in the depth dimension. The superimposition of image detail is then resolved by computing a 3D reconstruction of individual thick sections, using back-projection algorithms that are essentially the same as those used in medical imaging procedures such as magnetic resonance imaging (MRI) and computerized axial tomography (CAT). Briefly, a projection image records the amount of mass density encountered by imaging rays, as illustrated in Fig 1A. Because the path of the imaging rays is known (or can be computed), the only unknown is how specimen mass was distributed along the imaging path (i.e., one dimension is always "lost" during projection imaging). The approach of the back-projection algorithms is to simply distribute the known specimen mass evenly over computed back-projection rays that in effect retrace the path of the imaging rays. In this way, specimen mass is projected back into a reconstruction volume (i.e., "back-projected"). When this process is repeated for a series of projection images recorded from different tilt angles, back-projection rays from the different images intersect and reinforce one another at the points where mass is found in the original structure, as illustrated in Fig 1B. Hence, the 3D mass of the specimen is reconstructed from a series of 2D images. For a more in-depth description of imaging and tomographic reconstruction, see
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Data Collection |
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Data collection for electron tomography is accomplished by tilting the specimen in the electron beam. The resolution and quality of the reconstruction are directly dependent on how finely spaced the tilt images are and over how wide an angular range they extend. Therefore, the strategy is to collect as many tilt images over as wide an angular range as is feasible. Generally, this consists of collecting images at 12° angular intervals over an angular range of ±60 to 70°. Therefore, electron tomography requires that the electron microscope be equipped with an accurate tilt stage and a specimen holder that can accommodate reasonably high tilt angles. Although it is possible to collect a tomographic tilt series on a manual tilt stage, such a procedure is extremely tedious. Therefore, routine application of tomography requires a motorized tilt stage that is reasonably eucentric, and is equipped with a digital angular readout. These features are readily available on most modern electron microscopes.
Another feature that is becoming increasingly common on electron microscopes is a digital camera for image recording. Because the data must be in digital form for computation of the reconstruction, direct digital recording can be a considerable timesaver compared with film recording and subsequent digitization. Direct digital recording also obviates the need to interrupt data collection for film changes and processing. Because it is now recommended to use dual-axis data collection whenever feasible (see below), typical electron tomographic data sets range from 120 to 280 images. Furthermore, one usually wants to see more than one example of the 3D structure and is often interested in different functional states of the organelle and/or examples from different cell types. Therefore, individual studies can easily involve collecting thousands of images and data throughput becomes an important consideration. In such situations, image acquisition via a CCD camera can be an important boon. For many studies, an image size of 1024 x 1024 pixels is sufficient to ensure the requisite pixel resolution over an adequate area of the specimen. However, for studying large areas of a cell, montaging (i.e., pasting together CCD images from adjacent slightly overlapping CCD images) may be needed. This function is normally supplied along with the imaging software purchased with the CCD camera system. For these large specimen areas it is often preferable to go back to using film because film provides greater spatial resolution (i.e., many more pixels) than CCD cameras (e.g., see
Another important consideration for data collection is accelerating voltage. At 60° tilt, the path length of the electron beam through the specimen is twice the specimen thickness. At 70° it is three times the specimen thickness. Therefore, at conventional accelerating voltages image quality rapidly deteriorates at high tilt angles. For this reason, intermediate-voltage (200400 kV, IVEM), high-voltage (1.0 MV, HVEM), or energy-filtered electron microscopes are highly recommended for specimens thicker than 120150 nm. Although a few smaller specimens, such as negatively stained preparations of isolated chromatin fibers, have been successfully studied with electron tomography using conventional accelerating voltages, most applications, particularly those using thick plastic sections, have used IVEMs or HVEMs. If access to an IVEM or HVEM equipped with a proper tilt stage is a limitation, data can be collected at one of the three NIH Biotechnological Resources that are set up for electron tomography: at the University of Colorado at Boulder; at the University of California at San Diego; and at the Wadsworth Center in Albany, NY. In addition to having the proper microscope, tilt stage, and camera, the Resources also have appropriate software packages and experienced personnel who can assist the user.
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Data Processing |
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Because goniometers in electron microscopes are not perfectly eucentric (although some of the newer stages come extremely close), a certain amount of specimen tracking and refocus is necessary during collection of a tomographic tilt series. As a result, images of a tilt series must be translationally and rotationally aligned to bring them into register for computation of the 3D reconstruction (i.e., for the back-projection rays to intersect at centers of mass as illustrated in Fig 1B, the tilt images must be properly aligned). Alignment is most commonly accomplished by placing colloidal gold particles on the surface of the plastic section before collecting the tilt set. The positions of a given set of gold markers on each tilt image are then used by alignment algorithms to calculate shift vectors and rotations that bring the images into register (
Once the tilt images are aligned, the 3D reconstruction can be computed using a back-projection algorithm. Although this is the most sophisticated and computer-intensive part of the process, it is also the part that is most transparent to the user. In general, one simply submits a program command file with the aligned images and waits for the reconstruction.
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Visualizing the Reconstruction |
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After the 3D reconstruction is computed, the user is again faced with viewing a 3D object on a 2D medium such as the computer monitor. Often an experienced user can answer the biological questions at hand simply by viewing sequential thin 2D slices through the reconstructed volume. Although this is analogous to serial-section analysis, in tomography the thickness of the slices is determined by the pixel size chosen during digitization, and it can be as thin as 1 nm. Although viewing sequential slices frequently suffices for understanding simpler systems, 3D visualization is usually required for analysis of more complex organelles, communication of results to others, and quantitative measurements. The most common approach is to use some form of segmentation to extract features of interest from the volume and then to view the subvolume using surface- or volume-rendering methods (reviewed by
Fig 2 shows an example of volume segmentation applied to mitochondria. Membranes of individual cristae were traced using Sterecon, a software package developed in our laboratory for tracing 2D and 3D contours on serial sections and in 3D volumes (
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Fig 3 illustrates the segmentation procedure being used to measure microtubule penetration into kinetochores from mammalian chromosomes. These data were collected to test a model predicting that the direction of kinetochore motion would be correlated with the depth of microtubule penetration into the outer plate (see
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Limitations and Special Considerations |
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Limited Tilt Range
Because the planar dimensions of a standard TEM specimen grid are three orders of magnitude larger than its thickness, the effective electron beam path through the specimen rapidly increases for tilt angles above 60° (see illustration in
Specimen Thinning
Another limitation is due to a 2550% thinning of plastic sections that occurs on exposure to the electron beam (
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Resolution and Choosing Between Electron Tomography and Serial Section 3D Reconstruction |
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The resolution of a tomographic reconstruction is dependent upon the size of the specimen and the number of tilt views collected (
For plastic sections, the nominal resolution for a given tilt angle interval is dependent on the section thickness and maximal tilt angle rather than the actual size of the reconstructed specimen (
where d is resolution, a the angular interval (in degrees), T the section thickness, and max the maximal tilt angle in the series. Using this formulation, the resolution for a tomographic reconstruction of a specimen from a 0.250-µm-thick section is 8.7 nm if the tilt angle interval is 1° and the maximal tilt angle is 60°.
As mentioned above, the resolution is not isotropic. The above formula gives the resolution in planes perpendicular to the electron beam, represented in a tomographic reconstruction by slices parallel to the surface of the section. Resolution in planes (slices) perpendicular to the section surface is more strongly influenced by the elongation factor resulting from missing angular information. This factor decreases the resolution in the vertical (depth) direction in these slices by at least 20%. In the case of single-axis tilting, resolution in slices perpendicular to the tilt axis is further decreased by streaking artifacts that cause linear elements to disappear from view. These artifacts are barely noticeable, however, if the dual-axis tilting method is used (
The easiest way to improve resolution is to cut thinner sections, as long as the section is thick enough to provide useful depth information in the 3D reconstruction. Previously, we emphasized that resolution limit is not synonymous with detection limit (
The resolution of serial-section reconstructions is limited to twice the section thickness (see
There are many applications for which serial-section analysis is adequate or even preferable to tomographic reconstruction. Hence, it is advisable to consider both approaches in the context of the goals of the study. Taking an example from our own work, it is easier to study the structure of an entire kinetochore using serial-section analysis because it is difficult to ensure that the whole structure will be contained within a single thick section. If the section is cut thick enough (several times thicker than the kinetochore) to ensure that the entire structure is contained, the tomographic resolution would be inadequate. Therefore, when we wanted to find out how many microtubules were bound to kinetochores under different conditions, we used serial-section analysis to ensure that we did not miss part of the kinetochore (
Because tomography provides higher depth resolution and serial-section reconstruction is able to track complete structures over relatively large distances, it is natural to try to combine the two techniques. Serial-section tomography is in fact feasible (
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Examples of Recent Applications |
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The number of structural determinations employing electron tomography has grown rapidly over the past decade. A representative (but not exhaustive) list of electron tomographic applications is presented in Table 1. In the following paragraphs we describe five of these applications in more depth, with the intent of providing an indication of the scope of electron tomography and the kinds of questions that can be answered using this technique.
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The key step in bone formation is deposition of calcium phosphate crystals into an extracellular matrix composed chiefly of Type I collagen. Electron tomography of initial crystal growth provided the first direct 3D imaging of the size, shape, orientation, and growth of crystals in normal mineralizing tissue (
An early controversy developed as to the structure of mitochondrial inner membrane folds known as cristae (reviewed in 30 nm) tubular connections (
The 3D reconstruction of a Golgi apparatus by
In many cell types, microtubule growth is nucleated from the amorphous region of centrosomes known as the pericentriolar material. Nucleation requires -tubulin, a tubulin protein found in centrosomes that localizes to the minus ends of microtubules (
-tubulin and other protein components have been isolated from Xenopus egg extracts (
-tubulin ring complexes (
TuRcs) will nucleate microtubule assembly in vitro. Electron tomography demonstrated that
-tubulin containing rings of similar size is located in the pericentriolar material of isolated Drosophila centrosomes (
TuRcs are composed of 12 columnar subunits that are paired and arranged into a single turn of a helix (
TuRc. Electron tomography also demonstrated that
TuRc structures are located at the minus ends of microtubules nucleated by purified
TuRcs or isolated centrosomes. From these data, the authors extended the template model for microtubule initiation by proposing that a
-tubulin located in each of the 12 columnar subunits of the
TuRc initiates growth of a single microtubule protofilament. The thirteenth protofilament is initiated via lateral interactions with the twelfth, and possibly the first, protofilament. Electron tomography has also played a central role in similar studies to elucidate microtubule nucleation via spindle pole bodies in yeast (
In a final example, electron tomography was combined with time-resolved electron microscopy and limited spatial averaging to directly visualize the motor actions of myosin in insect flight muscle (
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Future Directions |
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At present, much of the technical development in electron tomography is focused on perfecting its application to frozenhydrated specimens. This is important because frozenhydrated specimens do not require chemical fixation, dehydration, or staining, and thus represent the specimen in a near-native state (
A viable alternative to cryoultramicrotomy for eukaryotic cell cultures and tissue specimens is rapid freezing followed by freeze-substitution with an organic solvent. The specimen can then be embedded in plastic (reviewed by
Other developments that will improve the quality of tomographic reconstruction from plastic sections include the following: minimizing specimen thinning by means of low-temperature imaging and optimization electron exposure (
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Conclusions |
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Electron tomography is a method for 3D ultrastructural determination that has proven its worth in a variety of biological studies. It is best applied to structures in the 501000-nm size range, where it fills the gap between higher-resolution studies on isolated macromolecules and larger-scale studies on cells and tissue. Improvements in microscope instrumentation, computing power, and software have made it possible to implement electron tomography in a reasonably well-equipped EM laboratory. The majority of tomographic studies are carried out on sections of plastic-embedded material, but a few tomographic reconstructions of frozenhydrated material have also been published. Further improvements in imaging techniques and reconstruction software promise to increase the quality and breadth of electron tomographic reconstructions.
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Acknowledgments |
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BFM is supported by NSF Grant MCB 9808879 and the NIH/NCRR Biomedical Research Technology Program Grant RR01219, which funds Wadsworth Center's Resource for Visualization of Biological Complexity as a National Biotechnological Resource Center. MM receives support from the State of New York through the Wadsworth Center.
We thank Dr William Samsonoff for helpful comments on the manuscript and Drs Carmen Mannella, Joachim Frank, Pawel Penczek, and Conly Rieder for many stimulating conversations. Much of the experimental work illustrated in this review used facilities maintained by the Resource for Visualization of Biological Complexity, and by the Wadsworth Center's core facilities for electron microscopy and video light microscopy.
Received for publication August 15, 2000; accepted November 27, 2000.
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