Three-Dimensional Structure of Neurospora Mitochondria: New Insights Provided by Electron Tomography of the Frozen-Hydrated Organelles

1999 ◽  
Vol 5 (S2) ◽  
pp. 452-453
Author(s):  
D. Nicastro ◽  
A. S. Frangakis ◽  
S. Nickell ◽  
W. Baumeister
Keyword(s):  
Low Dose ◽  
Functional Organization ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Dimensional Structure ◽  
Data Sets ◽  
Cell Organelles ◽  
Energy Filtering ◽  
Tilt Series

Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context.In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).

Crystallographic Reconstruction of the Acrosomal Process from Limulus Polyphemus Sperm

1998 ◽  
Vol 4 (S2) ◽  
pp. 460-461
Author(s):  
M. B. Sherman ◽  
J. Jakana ◽  
S. Sun ◽  
P. Matsudaira ◽  
W. Chiu ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Horseshoe Crab ◽  
Carbon Film ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Limulus Polyphemus ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Diffraction Mode ◽  
Tilt Series

The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice2 and is up to 60 (im long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. The goal of the current study is to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope3 to reconstruct its three-dimensional structure without assuming helical symmetry.The acrosomal process was purified as described.34 Bundles were embedded in vitreous ice over holes on a holey carbon film on copper grids. The specimen was kept at -167°C in a JEOL 4000EX electron microscope operating at 400 kV. Straight 6-10 (im long bundles were found using a TV-rate CCD camera in defocused diffraction mode.

Computer-aided methods for 3-D visualization of serial sections and thick biological specimens

1992 ◽  
Vol 50 (2) ◽  
pp. 1060-1061
Author(s):  
Mark Ellisman ◽  
Maryann Martone ◽  
Gabriel Soto ◽  
Eleizer Masliah ◽  
David Hessler ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Three Dimensional ◽  
Neuritic Plaque ◽  
Dimensional Structure ◽  
Data Sets ◽  
Molecular Physiology ◽  
Research Activities ◽  
Computer Aided ◽  
Dimensional Reconstruction

Structurally-oriented biologists examine cells, tissues, organelles and macromolecules in order to gain insight into cellular and molecular physiology by relating structure to function. The understanding of these structures can be greatly enhanced by the use of techniques for the visualization and quantitative analysis of three-dimensional structure. Three projects from current research activities will be presented in order to illustrate both the present capabilities of computer aided techniques as well as their limitations and future possibilities.The first project concerns the three-dimensional reconstruction of the neuritic plaques found in the brains of patients with Alzheimer's disease. We have developed a software package “Synu” for investigation of 3D data sets which has been used in conjunction with laser confocal light microscopy to study the structure of the neuritic plaque. Tissue sections of autopsy samples from patients with Alzheimer's disease were double-labeled for tau, a cytoskeletal marker for abnormal neurites, and synaptophysin, a marker of presynaptic terminals.

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

scholarly journals High-resolution three-dimensional probes of biomaterials and their interfaces

2012 ◽  
Vol 370 (1963) ◽  
pp. 1337-1351 ◽  
Author(s):  
Kathryn Grandfield ◽  
Anders Palmquist ◽  
Håkan Engqvist
Keyword(s):  
High Resolution ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Controlled Drug Release ◽  
Biological Tissues ◽  
Dimensional Structure ◽  
Bone Interface ◽  
Biomaterial Interfaces ◽  
Titania Coating

Interfacial relationships between biomaterials and tissues strongly influence the success of implant materials and their long-term functionality. Owing to the inhomogeneity of biological tissues at an interface, in particular bone tissue, two-dimensional images often lack detail on the interfacial morphological complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar length scale. Electron tomography (ET) can meet these challenges by enabling high-resolution three-dimensional imaging of biomaterial interfaces. In this article, we review the fundamentals of ET and highlight its recent applications in probing the three-dimensional structure of bioceramics and their interfaces, with particular focus on the hydroxyapatite–bone interface, titanium dioxide–bone interface and a mesoporous titania coating for controlled drug release.

Televisualization: An Aid To Collaborative Research In Molecular Structure Biology

1998 ◽  
Vol 4 (S2) ◽  
pp. 32-33
Author(s):  
M. F. Schmid ◽  
P. Matsudaira ◽  
M. T. Dougherty ◽  
M. B. Sherman ◽  
C. Henn ◽  
...  
Keyword(s):  
Image Processing ◽  
Actin Filaments ◽  
Collaborative Research ◽  
Horseshoe Crab ◽  
Hexagonal Lattice ◽  
Three Dimensional ◽  
Limulus Polyphemus ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Tilt Series

Collaboration between local microscopists and image processing specialists, and their remote biological colleagues, has been hampered by the difficulty of i) transferring the three-dimensional reconstructions of macromolecules resulting from the cryomicroscopy and image processing, ii) viewing the results in a meaningful way, and iii) communicating the results and the interpretations derived therefrom to each other.The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice and is up to 60 μm long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. Our goal was to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope to reconstruct its three-dimensional structure at 45 Å resolution.

Three-Dimensional Imaging of In Situ Specimens with Low-Dose Electron Tomography to Analyze Protein Conformation

2004 ◽  
Vol 2 (5) ◽  
pp. 561-567 ◽  
Author(s):  
Martina Banyay ◽  
Fredrik Gilstring ◽  
Elenor Hauzenberger ◽  
Lars-Göran Öfverstedt ◽  
Anders B. Eriksson ◽  
...  
Keyword(s):  
Protein Conformation ◽  
Low Dose ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Three Dimensional Imaging

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Cryo-Electron Tomography Reveals the Comparative Three-Dimensional Architecture of Prochlorococcus, a Globally Important Marine Cyanobacterium

2007 ◽  
Vol 189 (12) ◽  
pp. 4485-4493 ◽  
Author(s):  
Claire S. Ting ◽  
Chyongere Hsieh ◽  
Sesh Sundararaman ◽  
Carmen Mannella ◽  
Michael Marko
Keyword(s):  
Cell Wall ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane System ◽  
Niche Differentiation ◽  
Dimensional Structure ◽  
Comparative Genomic ◽  
Photosynthetic Membrane ◽  
Peptidoglycan Biosynthesis ◽  
Cryo Electron Tomography

ABSTRACT In an age of comparative microbial genomics, knowledge of the near-native architecture of microorganisms is essential for achieving an integrative understanding of physiology and function. We characterized and compared the three-dimensional architecture of the ecologically important cyanobacterium Prochlorococcus in a near-native state using cryo-electron tomography and found that closely related strains have diverged substantially in cellular organization and structure. By visualizing native, hydrated structures within cells, we discovered that the MED4 strain, which possesses one of the smallest genomes (1.66 Mbp) of any known photosynthetic organism, has evolved a comparatively streamlined cellular architecture. This strain possesses a smaller cell volume, an attenuated cell wall, and less extensive intracytoplasmic (photosynthetic) membrane system compared to the more deeply branched MIT9313 strain. Comparative genomic analyses indicate that differences have evolved in key structural genes, including those encoding enzymes involved in cell wall peptidoglycan biosynthesis. Although both strains possess carboxysomes that are polygonal and cluster in the central cytoplasm, the carboxysomes of MED4 are smaller. A streamlined cellular structure could be advantageous to microorganisms thriving in the low-nutrient conditions characteristic of large regions of the open ocean and thus have consequences for ecological niche differentiation. Through cryo-electron tomography we visualized, for the first time, the three-dimensional structure of the extensive network of photosynthetic lamellae within Prochlorococcus and the potential pathways for intracellular and intermembrane movement of molecules. Comparative information on the near-native structure of microorganisms is an important and necessary component of exploring microbial diversity and understanding its consequences for function and ecology.

A Novel Method for Automated Acquisition of Tilt Series for Electron Tomography Based on Pre-Calibration of the Specimen Stage

2000 ◽  
Vol 6 (S2) ◽  
pp. 1148-1149
Author(s):  
U. Ziese ◽  
A.H. Janssen ◽  
T.P. van der Krift ◽  
A.G. van Balen ◽  
W.J. de Ruijter ◽  
...  
Keyword(s):  
High Resolution ◽  
3D Reconstruction ◽  
Cell Biology ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Imaging Method ◽  
3D Images ◽  
Tilt Series ◽  
3D Information ◽  
Structural Arrangements

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. Conventional TEM images are in first approximation mere 2D-projections of a 3D sample under investigation. With electron tomographya series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments (so called tilt-series). For the subsequent 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 true 3D information with nm-scale resolution of individual and unique samples. For (cell) biology and material science applications the availability of high-resolution 3D images of structural arrangements within individual samples provides unique architectural information that cannot be obtained otherwise. Routine application of electron tomography will comprise a major revolutionary step forward in the characterization of complex materials and cellular arrangements.

Automation of Tilt Series Acquisition in Transmission Electron Microscopy for Applications in Biology and Materials Science

2001 ◽  
Vol 7 (S2) ◽  
pp. 88-89
Author(s):  
Ingo Daberkow ◽  
Bernhard Feja ◽  
Peter Sparlinek ◽  
Hans R. Tietz
Keyword(s):  
3D Reconstruction ◽  
Data Acquisition ◽  
San Francisco ◽  
High Speed ◽  
Materials Science ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Max Planck ◽  
Automatic Data ◽  
Tilt Series

During the last decade, computation of a three-dimensional image from a tilt series (3D reconstruction) has become a well established method, of which a variety of implementations are available. The term “electron tomography” is now generally used for this type of data acquisition and 3D reconstruction. An overview over the techniques involved is given in.With the introduction of micro-processor-controlled TEMs and cooled slow-scan CCD cameras and with the progress in performance of high-speed computers, automation of complex imaging procedures became mainly a task of developing appropriate software, using the control facilities of the microscope. in this way, automated electron tomography was realized in 1990 at the Max- Planck-Institute for Biochemistry in Martinsried, and at about the same time at the University of California in San Francisco (UCSF). New techniques for automatic focusing and alignment, developed somewhat earlier , have been integrated in these automated tomography procedures. in the following we discuss the requirements of automatic data acquisition and the present implementation for several TEMs.

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