Approaches for High Resolution Cryo-Electron Tomography of Biological Specimens

1997 ◽  
Vol 3 (S2) ◽  
pp. 1111-1112
Author(s):  
D.A. Agard ◽  
M.B. Braunfeld ◽  
Hans Chen ◽  
Rebecca McQuitty ◽  
John Sedat
Keyword(s):  
Data Collection ◽  
Structural Information ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Electron Microscopic ◽  
Subcellular Organelles ◽  
Voltage Electron ◽  
Cell Components ◽  
Electron Microscopes ◽  
Biological Complexes

Electron tomography is a powerful tool for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles. Use of intermediate voltage electron microscopes extended the technique by providing the means to examine very large and non-symmetrical subcellular organelles, at resolutions beyond what would be possible using light microscopy. Recent studies using electron tomography on a variety cellular organelles and assemblies such as centrosomes (Moritz et al.,1995a,b), kinetochores (McEwen, 1993) and chromatin (Woodcock, 1994), have clearly demonstrated the power of this method for obtaining 3D structural information on non-symmetric cell components. When combined with biochemical and molecular observations, these 3D reconstructions have provided significant new insights into biological function.Although the information that tomography provides is unique, its use as a general tool in the biological community has been limited due to the complexities involved in data collection and processing.We are simultaneously trying to make this approach accessible through automation as well as trying to extend the resolution and accuracy of the reconstructions. Significant, has been the use of low-dose cryo-electron microscopic automated data collection methods.

Automated data collection for electron tomography

1992 ◽  
Vol 50 (2) ◽  
pp. 1044-1045
Author(s):  
D.A. Agard ◽  
A.J. Koster ◽  
M.B. Braunfeld ◽  
J.W. Sedat
Keyword(s):  
Data Collection ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Higher Order ◽  
Projection Data ◽  
Voltage Electron ◽  
Digital Format ◽  
Central Interest ◽  
Focus Variation

Three-dimensional imaging has become an important addition to the variety of methods available for research on biological structures. Non-crystalline samples can be examined by high resolution electron tomography which requires that projection data be collected over a large range of specimen tilts. Practical limitations of tomography are set by the large number of micrographs to be processed, and by the required (and tedious) recentering and refocusing of the object during data collection; especially for dose sensitive specimens. With automated electron tomography a number of these problems can be overcome. First, the images are recorded directly in digital format, using a cooled slow scan CCD camera, and, with automatic tracking and correction for image shift and focus variation, a pre-aligned dataset is obtained, with every image recorded under well defined imaging conditions.At UCSF, we use intermediate voltage electron tomography to study higher-order chromatin structure. Of central interest is elucidating the higher-order arrangement of the 30nm chromatin fiber within condensed chromosomes through several phases of the cell cycle and, in collaboration with Chris Woodcock, the structure of the 30 nm fiber.

Tomographic methods for detailed examination of large structures in the nervous system

1993 ◽  
Vol 51 ◽  
pp. 96-97
Author(s):  
Gabriel E. Soto ◽  
Stephen J. Young ◽  
Maryann E. Martone ◽  
Thomas J. Deerinck ◽  
Stephan Lamont ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Detailed Examination ◽  
Thick Section ◽  
Section Thickness ◽  
Serial Sectioning ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Large Structures ◽  
Electron Microscopes ◽  
Axial Tilt

One of the limitations of electron microscopy has been the requirement for very thin samples to allow penetration of the electron beam. It is often the case that structures of interest are not contained within a single thin section. In these cases, serial sectioning techniques are required to reconstruct the object in its entirety. The use of higher voltage electron microscopes has allowed researchers to examine specimens up to fifty times thicker than those suitable for a conventional TEM. However, images from thick sections are often difficult to interpret as the electron micrograph is essentially a projection of the overlapping material within the section. The method of computerized axial tilt electron microscopic tomography offers the potential to visualize and analyze information contained in a thick section by deriving a three dimensional volume from a series of projections acquired by collecting images of the specimen at successive tilt increments about the Y axis. Unfortunately there are practical limitations to the resolution that can be obtained using this technique with very thick sections. Resolution of the tomogram increases with finer tilt sampling and an increased range of tilts but decreases with section thickness.

Properties and dose dependence of embedding media for cryo-automated electron tomography

1993 ◽  
Vol 51 ◽  
pp. 436-437
Author(s):  
M.B. Braunfeld ◽  
A.J. Koster ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Electron Tomography ◽  
Dose Dependence ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
High Doses ◽  
Beam Dose ◽  
Stability And Consistency ◽  
Imaging Conditions

Electron tomography is well suited to the study of complicated, non symmetric biological structures. In our laboratory, we use intermediate voltage electron microscopic tomography to follow complex paths of chromatin fibers within intact sections of Hela telophase chromosomes. In order to accurately reconstruct these features at resolutions beyond 50Å, precise imaging conditions and data collection schemes have been developed and employed.To obtain useful high resolution information, the specimen needs to be well preserved. Data collection must also be accurate and self-consistent. However, a serious limitation has been radiation damage to the specimen during scanning, and data collection. Because of the high doses required for tomography, the standard approach has been to accept the inevitability of serious radiation damage, and to heavily pre-irradiate the sample in an attempt to provide stability and consistency during data collection.The use of fully-automated data collection methods allows a substantial decrease in beam dose, suggesting that the entire approach should be reevaluated.

Increasing Access to Tomographic Resources: Web-based Telemicroscopy and Database

2001 ◽  
Vol 7 (S2) ◽  
pp. 92-93
Author(s):  
M. E. Martone ◽  
S. Peltier ◽  
S. Lamont ◽  
A. Gupta ◽  
B. Ludaescher ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
High Voltage ◽  
Cell Biology ◽  
Electron Tomography ◽  
The United States ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Electron Microscopes

The application of electron tomography to cell biology has led to important insights into the 3D fine structure of subcellular processes and organelles. Tomography has been particularly useful for studying relatively large, multi-component structures such as the Golgi apparatus, mitochondria and synaptic complexes. When combined with very powerful high voltage electron microscopes, tomography has also provided high resolution quantitative views of extended structures such as neuronal dendrites in very thick sections (4 μm) at electron microscopic resolution. The utility of tomography is twofold: first, it provides 3D examination of subcellular structure without the need for serial section analysis; second, because the computed slices through the tomographic volumes can be much thinner than is possible to produce by physical sectioning, it reveals structural detail in the range of 5-30 nm that tends to be obscured in conventional thin sections. Tomographic analysis has forced re-assessment of long-standing views of organelles such as mitochondria and the Golgi apparatus and as the technique advances, additional insights are likely forthcoming.Electron tomography is an expensive technique, both in terms of the instruments used and the computational resources required. The three major high voltage electron microscope resources in the United States, San Diego, Boulder and Albany, all are actively engaged in tomographic research and offer this important technology to the scientific community at large.

scholarly journals Three-Dimensional Macromolecular Organization of Cryofixed Myxococcus xanthus Biofilms as Revealed by Electron Microscopic Tomography

2009 ◽  
Vol 191 (7) ◽  
pp. 2077-2082 ◽  
Author(s):  
Hildur Palsdottir ◽  
Jonathan P. Remis ◽  
Christoph Schaudinn ◽  
Eileen O'Toole ◽  
Renate Lux ◽  
...  
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
3D Visualization ◽  
Myxococcus Xanthus ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Ultrastructure ◽  
Specimen Preparation ◽  
Electron Microscopic ◽  
Cytoplasmic Organization

ABSTRACT Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.

Automated electron tomography: from data collection to image processing

1995 ◽  
Vol 53 ◽  
pp. 26-27
Author(s):  
Weiping Liu ◽  
Jennifer Fung ◽  
W.J. de Ruijter ◽  
Hans Chen ◽  
John W. Sedat ◽  
...  
Keyword(s):  
Image Processing ◽  
Data Collection ◽  
Structural Information ◽  
Imaging Technique ◽  
Electron Tomography ◽  
Ccd Camera ◽  
Automated Data Collection ◽  
Full Control ◽  
Transmission Electron ◽  
Amorphous Structures

Electron tomography is a technique where many projections of an object are collected from the transmission electron microscope (TEM), and are then used to reconstruct the object in its entirety, allowing internal structure to be viewed. As vital as is the 3-D structural information and with no other 3-D imaging technique to compete in its resolution range, electron tomography of amorphous structures has been exercised only sporadically over the last ten years. Its general lack of popularity can be attributed to the tediousness of the entire process starting from the data collection, image processing for reconstruction, and extending to the 3-D image analysis. We have been investing effort to automate all aspects of electron tomography. Our systems of data collection and tomographic image processing will be briefly described.To date, we have developed a second generation automated data collection system based on an SGI workstation (Fig. 1) (The previous version used a micro VAX). The computer takes full control of the microscope operations with its graphical menu driven environment. This is made possible by the direct digital recording of images using the CCD camera.

EMCAT: An automatic image-processing system for electron-microscopic tomography

1994 ◽  
Vol 52 ◽  
pp. 418-419
Author(s):  
Weiping Liu ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Image Processing ◽  
Structural Information ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Processing System ◽  
Electron Microscopic ◽  
Data Set ◽  
Ordered Structures ◽  
Automatic Image Processing ◽  
Em Tomography

Any real world object is three-dimensional. The principle of tomography, which reconstructs the 3-D structure of an object from its 2-D projections of different view angles has found application in many disciplines. Electron Microscopic (EM) tomography on non-ordered structures (e.g., subcellular structures in biology and non-crystalline structures in material science) has been exercised sporadically in the last twenty years or so. As vital as is the 3-D structural information and with no existing alternative 3-D imaging technique to compete in its high resolution range, the technique to date remains the kingdom of a brave few. Its tedious tasks have been preventing it from being a routine tool. One keyword in promoting its popularity is automation: The data collection has been automated in our lab, which can routinely yield a data set of over 100 projections in the matter of a few hours. Now the image processing part is also automated. Such automations finish the job easier, faster and better.

The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis

1993 ◽  
Vol 106 (1) ◽  
pp. 261-274 ◽  
Author(s):  
M.W. Goldberg ◽  
T.D. Allen
Keyword(s):  
Nuclear Pore Complex ◽  
Three Dimensional ◽  
Underlying Structure ◽  
Nuclear Pore ◽  
Electron Microscopic ◽  
Biologically Relevant ◽  
Fine Grained ◽  
Outer Periphery ◽  
Electron Microscopes ◽  
Scanning Electron

The structure of the nuclear pore complex (NPC) has been previously studied by many different electron microscopic techniques. Recently, scanning electron microscopes have been developed that can visualise biologically relevant structural detail at the same level of resolution as transmission electron microscopes and have been used to study NPC structure. We have used such an instrument to visualise directly the structure of both cytoplasmic and nucleoplasmic surfaces of the NPC of manually isolated amphibian oocyte nuclear envelopes that have been spread, fixed, critical point dried and coated with a thin fine-grained film of chromium or tantalum. We present images that directly show features of the NPC that are visible at each surface, including coaxial rings, cytoplasmic particles, plug/spoke complexes and the nucleoplasmic basket or fishtrap. Some cytoplasmic particles are rod-shaped or possibly “T”-shaped, can be quite long structures extending into the cytoplasm and may be joined to the coaxial ring at a position between each subunit. Both coaxial rings, which are proud of the membranes, can be exposed by light proteolytic digestion, revealing eight equal subunits each of which may be bipartite. We have determined that the nucleoplasmic filaments that make up the baskets are attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring. The space left between adjacent basket filaments would exclude particles bigger than about 25 nm, which is consistent with the exclusion limit previously found for NPC-transported molecules.

Three-Dimensional Analysis of Tranverse Tubules in Normal and Failing Heart: A Combined Confocal and High Voltage Electron Microscope Study

1997 ◽  
Vol 3 (S2) ◽  
pp. 231-232
Author(s):  
M. E. Martone ◽  
V. M. Edelman ◽  
A. Thor ◽  
S. J. Young ◽  
S. P. Lamont ◽  
...  
Keyword(s):  
High Voltage ◽  
Three Dimensional ◽  
Cardiac Cells ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Spontaneously Hypertensive ◽  
3 Dimensional ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
T Tubules

Early electron microscopic studies documented that significant changes in the membrane systems of cardiac cells occur in both ischemic and non-ischemic heart failure. These studies relied on analysis of two-dimensional sections and although quantitative changes were observed, the overall organization of the tranverse tubules (T-tubules) and the sarcoplasmic reticulum could not be assessed. In a 3-dimensional study using high voltage electron microscopy (EM) of the T-tubules in spontaneously hypertensive rats, Nakamura and Hama (1991) observed that concomitant with an increase in surface area, the T-tubule system becomes progressively more disorganized and exhibits structural irregularities such as increased numbers of longitudinal tubules, numerous short dead end branches and complex tubular aggregates. These authors suggested that this disorganization may interfere with synchronous contraction over the entire cell.In the present study, we examined the 3-dimensional organization of T-tubules in the left ventricle of explanted human hearts using confocal microscopy and EM tomography.

Correlated 3D Light and Electron Microscopy of Large, Complex Structures: Analysis of Transverse Tubules in Heart Failure

1998 ◽  
Vol 4 (S2) ◽  
pp. 440-441
Author(s):  
Maryann E. Martone ◽  
Andrea Thor ◽  
Stephen J. Young ◽  
Mark H. Ellisman.
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Structural Features ◽  
Electron Microscopic Level ◽  
Specimen Preparation ◽  
Large Sample Size ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

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