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.

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.

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.

A Novel Method of Data Collection for Automated Electron Tomography Based upon Pre-cal1bration of Image Shifts and Defocus Changes

2001 ◽  
Vol 7 (S2) ◽  
pp. 78-79
Author(s):  
Ulrike Ziese ◽  
Ries Janssen ◽  
Willie Geerts ◽  
Theo van der Krift ◽  
Auke van Balen ◽  
...  
Keyword(s):  
Data Collection ◽  
3D Reconstruction ◽  
Electron Tomography ◽  
Ccd Camera ◽  
Current Status ◽  
Imaging Method ◽  
Major Step ◽  
Experimental Conditions ◽  
Digital Format ◽  
Tilt Series

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments. For the 3D-reconstruction, the images of the tilt series are aligned relative to each other and the 3D-reconstruction is computed. Electron tomography is the only technique that can provide 3D information with nm-scale resolution of individual and unique samples. Routine application of electron tomography will comprise a major step forward in the characterization of complex materials and cellular arrangements. When collecting tilt series for electron tomography image shifts and defocus changes have to be corrected for by the human operator. The repetitive correction of these changes is highly time consuming, error prone and very hard to carry out under low-dose imaging conditions.Many practical problems are overcome when electron tomography data collection is performed in an automated fashion. Automation includes the (a) image acquisition on a (digital) CCD camera, which implies that (b) changes in image position and defocus can be detected by on-line image processing and (c) immediately be corrected for by computer control of the microscope, (d) Finally, tilt series are directly available in digital format for subsequent processing. Typically, carrying out such an experiment would take a day, and the actual data collection 2-4 hours. in spite of the enormous progress made in terms of data collection speed during the last few years, the current status of automated tomography still does not meet the requirements that would make it a routinely applicable tool. For a great number of biological assays and research projects, results obtained under different experimental conditions have to be compared, and series of experiments have to be carried out. Therefore, we propose a novel approach for recording a tilt series that significantly increases data collection speed, and widens the applicability of the technique.

Automated electron tomography and CCD spot-scan imaging of biological macromolecules

1994 ◽  
Vol 52 ◽  
pp. 120-121
Author(s):  
A. J. Koster ◽  
J. Walz ◽  
U. Ziese ◽  
A. Stoschek ◽  
R. Hegerl ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Computer Control ◽  
3D Structure ◽  
Projection Data ◽  
Biological Macromolecules ◽  
Data Sets ◽  
Digital Format ◽  
Mode Beam ◽  
Set Up ◽  
2D Data

Computer control of a transmission electron microscope (TEM) and digital recording of images have shown to be useful tools in recording 2D data sets of macromolecules and larger cellular structures with accurately defined imaging conditions.3D structure of individual molecules can be obtained by electron tomography. The method requires that projection data are collected over a large range of specimen tilts. With automated electron tomography images are recorded in digital format, and lateral specimen displacement together with focus changes are automatically compensated for. Typically, with our experimental set-up, we collect 75 projections of negatively stained preparations over ±75° and 30 projections of ice-embedded molecules over ±60° with a total dose of 20-50 e/Å2.By using the spot-scan imaging mode beam-induced movements of ice-embedded specimens are greatly reduced, thus largely avoiding the loss of resolution associated with specimen instability. With CCD spot-scan imaging the size of the spot is matched to the CCD chip (Fig. 1).

Highlights of Selected Microscopy Research Resource Activities at San Diego

1997 ◽  
Vol 3 (S2) ◽  
pp. 275-276
Author(s):  
Mark H. Ellisman ◽  
Stephen J. Young ◽  
G. Y. Fan ◽  
Guy Perkins ◽  
Steve Lamont ◽  
...  
Keyword(s):  
San Diego ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Iterative Algorithms ◽  
Computation Time ◽  
Reconstruction Algorithms ◽  
Voltage Electron ◽  
Massively Parallel Computers ◽  
Imaging Research ◽  
Computationally Intensive

The intermediate high-voltage electron microscope (IVEM) located at the National Center for Microscopy and Imaging Research at San Diego (NCMIR) can image relatively thick specimens that contain substantial three-dimensional (3-D) structure. Electron tomography is an important tool used at NCMIR for deriving 3-D cellular and subcellular structure from IVEM images. Reconstruction algorithms commonly used in electron tomography include weighted back projection, and iterative algebraic reconstruction techniques such as ART and SIRT. Improvements in reconstruction quality are possible using the iterative algorithms. Because these algorithms are computationally intensive, we have ported them to massively parallel computers at the San Diego Supercomputer Center, reducing the computation time over that required with workstation level machines.The quality of tomographic data for the 3-D reconstruction of biological structures is also being enhanced by NCMIR projects to improve the microscope. We have designed and constructed special electron optics and microscope control systems for the JEOL 4000EX.

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 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.

Angular Resolved Electron Spectroscopy with Parallel Recording

1990 ◽  
Vol 48 (2) ◽  
pp. 370-371
Author(s):  
Huang Min ◽  
P.S. Flora ◽  
C.J. Harland ◽  
J.A. Venables
Keyword(s):  
Electron Spectroscopy ◽  
Low Voltage ◽  
Solid Angle ◽  
Detection System ◽  
Voltage Electron ◽  
Cylindrical Mirror ◽  
Inner Radius ◽  
Channel Plate ◽  
And Performance ◽  
Type Detector

A cylindrical mirror analyser (CMA) has been built with a parallel recording detection system. It is being used for angular resolved electron spectroscopy (ARES) within a SEM. The CMA has been optimised for imaging applications; the inner cylinder contains a magnetically focused and scanned, 30kV, SEM electron-optical column. The CMA has a large inner radius (50.8mm) and a large collection solid angle (Ω > 1sterad). An energy resolution (ΔE/E) of 1-2% has been achieved. The design and performance of the combination SEM/CMA instrument has been described previously and the CMA and detector system has been used for low voltage electron spectroscopy. Here we discuss the use of the CMA for ARES and present some preliminary results.The CMA has been designed for an axis-to-ring focus and uses an annular type detector. This detector consists of a channel-plate/YAG/mirror assembly which is optically coupled to either a photomultiplier for spectroscopy or a TV camera for parallel detection.

On the Use of POCS for Restoring the “Missing Wedge” in Electron Tomography

1990 ◽  
Vol 48 (1) ◽  
pp. 520-521
Author(s):  
Neng-Yu Zhang ◽  
Bruce F. McEwen ◽  
Joachim Frank
Keyword(s):  
Function Space ◽  
Convex Sets ◽  
Electron Tomography ◽  
Spinal Ganglion ◽  
Fourier Space ◽  
Missing Information ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
Energy Bound ◽  
Spatial Boundedness

Reconstructions of asymmetric objects computed by electron tomography are distorted due to the absence of information, usually in an angular range from 60 to 90°, which produces a “missing wedge” in Fourier space. These distortions often interfere with the interpretation of results and thus limit biological ultrastructural information which can be obtained. We have attempted to use the Method of Projections Onto Convex Sets (POCS) for restoring the missing information. In POCS, use is made of the fact that known constraints such as positivity, spatial boundedness or an upper energy bound define convex sets in function space. Enforcement of such constraints takes place by iterating a sequence of function-space projections, starting from the original reconstruction, onto the convex sets, until a function in the intersection of all sets is found. First applications of this technique in the field of electron microscopy have been promising.To test POCS on experimental data, we have artificially reduced the range of an existing projection set of a selectively stained Golgi apparatus from ±60° to ±50°, and computed the reconstruction from the reduced set (51 projections). The specimen was prepared from a bull frog spinal ganglion as described by Lindsey and Ellisman and imaged in the high-voltage electron microscope.

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