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.

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.

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,

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.

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.

Resources for the Study of Cellular Structure by High Voltage Electron Tomography, Serial Thin Sectioning, Specific Labeling, and Image Analysis

1997 ◽  
Vol 3 (S2) ◽  
pp. 273-274
Author(s):  
David Mastronarde ◽  
James Kremer ◽  
Eileen O’Toole ◽  
Mary Morphew ◽  
Mark Ladinsky ◽  
...  
Keyword(s):  
High Voltage ◽  
Cultured Cells ◽  
Electron Tomography ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
High Quality ◽  
Isotropic Resolution ◽  
Voltage Electron ◽  
Tissue Specimens

We are working to improve methods for the study of cellular fine structure. Our approach is to advance each of the key steps in the preparation of specimens for EM: high quality fixation that will preserve both structure and antigenicity; methods for specific labeling; efficient acquisition of 3-D electron microscopic data; and software for 3-D reconstruction and display.Our work on high quality structure preservation has focused on methods for fast freezing and freeze substitution. Both plunge freezing of specimens grown on coated gold grids and high pressure freezing of either cultured cells or tissue specimens have yielded well preserved material. These samples are suitable for freeze substitution fixation with either anhydrous aldehydes in acetone at -90°C, for the preservation of antigens, or aldehydes, tannic acid, OsO4, and uranyl acetate for optimal preservation the structure.We have used a JEOL JEM-1,000 high voltage microscope to image sections about 250nm thick, employing a goniometer stage to perform dual axis tomography for 3-D reconstruction with approximately isotropic resolution at ∼7nm.

Design and performance of instrumentation for automated single-tilt-axis electron tomography

1993 ◽  
Vol 51 ◽  
pp. 448-449
Author(s):  
A.J. Koster ◽  
H. Chen ◽  
W. Clyborne ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Data Collection ◽  
Electron Tomography ◽  
Limiting Factors ◽  
Projection Data ◽  
Reconstruction Algorithms ◽  
Native Form ◽  
Voltage Electron ◽  
Digital Format ◽  
High Resolution Analysis ◽  
And Performance

One of the driving questions in our group is into understanding how chromosomes are constructed from fibers of DNA wrapped around histones in their native form. To permit high resolution analysis of these highly complex fibers, we use intermediate voltage electron tomography. To obtain 50Å resolutions, we incorporate new approaches to overcome the resolution limiting factors determined by specimen fixation and staining techniques, data collection and 3D reconstruction algorithms.With our present instrumentation we can automatically collect a series of projection data of large, radiation sensitive objects with only a minimum of manual operation, with high accuracy and consistency. The images are recorded directly in digital format to overcome the time consuming task of digitizing negatives. Furthermore, the system offers automated eucentricity setting, automatic tracking of image shifts, and automatic focusing during data collection. Highly reliable data collection is ensured by closely monitoring the variation in image shift, defocus, average image intensity, and exposure time throughout the tilt series.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Design of Sensitive Photographic Materials for the High-Voltage Electron Microscope

1975 ◽  
Vol 33 ◽  
pp. 156-157
Author(s):  
Murray Vernon King ◽  
Donald F. Parsons
Keyword(s):  
Electron Microscope ◽  
Radiation Damage ◽  
High Voltage ◽  
Radiation Sensitivity ◽  
Fast Electrons ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
High Voltage Electron ◽  
Biological Studies ◽  
Low Sensitivity

Effective application of the high-voltage electron microscope to a wide variety of biological studies has been restricted by the radiation sensitivity of biological systems. The problem of radiation damage has been recognized as a serious factor influencing the amount of information attainable from biological specimens in electron microscopy at conventional voltages around 100 kV. The problem proves to be even more severe at higher voltages around 1 MV. In this range, the problem is the relatively low sensitivity of the existing recording media, which entails inordinately long exposures that give rise to severe radiation damage. This low sensitivity arises from the small linear energy transfer for fast electrons. Few developable grains are created in the emulsion per electron, while most of the energy of the electrons is wasted in the film base.

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

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