scholarly journals Cryomesh™: A New Substrate for Cryo-Electron Microscopy

2010 ◽  
Vol 16 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Craig Yoshioka ◽  
Bridget Carragher ◽  
Clinton S. Potter
Keyword(s):  
Electron Microscopy ◽  
Semiconductor Industry ◽  
Three Dimensional ◽  
Quality Of Data ◽  
Temperature Conductivity ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Using Data ◽  
Collection Methods

AbstractHere we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

Methods to Monitor and Improve the Performance of Specimen Holders for Transmission Electron Cryomicroscopy

1996 ◽  
Vol 54 ◽  
pp. 454-455
Author(s):  
B. L. Armbruster ◽  
B. Kraus ◽  
M. Pan
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Electron Beam Irradiation ◽  
Beam Irradiation ◽  
Quality Of Data ◽  
Electron Cryomicroscopy ◽  
Thermal Equilibration ◽  
Transmission Electron ◽  
Electron Microscopes

One goal in electron microscopy of biological specimens is to improve the quality of data to equal the resolution capabilities of modem transmission electron microscopes. Radiation damage and beam- induced movement caused by charging of the sample, low image contrast at high resolution, and sensitivity to external vibration and drift in side entry specimen holders limit the effective resolution one can achieve. Several methods have been developed to address these limitations: cryomethods are widely employed to preserve and stabilize specimens against some of the adverse effects of the vacuum and electron beam irradiation, spot-scan imaging reduces charging and associated beam-induced movement, and energy-filtered imaging removes the “fog” caused by inelastic scattering of electrons which is particularly pronounced in thick specimens.Although most cryoholders can easily achieve a 3.4Å resolution specification, information perpendicular to the goniometer axis may be degraded due to vibration. Absolute drift after mechanical and thermal equilibration as well as drift after movement of a holder may cause loss of resolution in any direction.

3D Morphological Characterization of Carbon-Black Aggregates Using Transmission Electron Microscopy

1994 ◽  
Vol 67 (2) ◽  
pp. 280-287 ◽  
Author(s):  
Tyler C. Gruber ◽  
T. W. Zerda ◽  
Michel Gerspacher
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Carbon Black ◽  
Three Dimensional ◽  
Reconstruction Algorithm ◽  
Morphological Characterization ◽  
Three Dimensional Modeling ◽  
Transmission Electron ◽  
Best Fitting ◽  
Using Data

Abstract A three-dimensional modeling technique is used to characterize the structure of carbon-black aggregates. The relative positions of individual particles in aggregates are determined using transmission electron microscopy (TEM). Data are acquired from two-dimensional projections taken with the aggregates at two different orientations with respect to the electron beam. Computerized aggregate models are generated using data from TEM projections in our reconstruction algorithm. Inspection of these models shows that their projections very closely replicate the TEM micrographs. Quantitative analysis of the aggregate models reveals that aggregates generally exhibit anisotropy, in the form of a reduction of aggregate breadth, or “flatness,” in one direction. The flat sides tend to align preferentially, along the plane of the TEM sample grid. The dimensions for each aggregate with respect to its best-fitting plane of flatness are determined, and are related through a “flatness index.”

Influence of Plural Scattering on the Image Quality of Thick Amorphous Objects in Transmission Electron Microscopy

1978 ◽  
Vol 36 (3) ◽  
pp. 230-243 ◽  
Author(s):  
H. Rose
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Finite Size ◽  
Amorphous Solid ◽  
Solid Objects ◽  
Transmission Electron ◽  
Carbon Foils ◽  
Shortrange Order ◽  
Arbitrary Objects

The ultimate goal of electron microscopy is to elucidate the three-dimensional atomic structure of arbitrary objects. Objects are generally classified with respect to their inherent structural symmetries. The extreme cases of total order and total disorder are perfect crystals and entirely amorphous objects. In the former case the atoms are arranged in a periodic order; in the latter they are distributed at random. Neither perfect crystals nor completely amorphous solid objects exist in reality. The finite size of the atoms gives rise in every solid amorphous object to a certain shortrange order that rules out complete disorder. The amorphous carbon foils commonly used as supporting films are an example of this behavior.

scholarly journals Automated, Robotic Preparation of Vitrified Samples for 2D and 3D Cryo Electron Microscopy

2005 ◽  
Vol 13 (6) ◽  
pp. 32-39 ◽  
Author(s):  
Peter. M. Frederik ◽  
Marc M.H. Storms
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Three Dimensional ◽  
Nano Technology ◽  
Biochemical Processes ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Starting Point ◽  
Fundamental Research ◽  
2D And 3D

Fundamental research within the scope of cell and structural biology as well as nano-technology is increasingly focusing on unraveling interactive biological and biochemical processes and pathways at the macromolecular level. For this, high resolution transmission electron microscopy (TEM) is indispensable. Of paramount importance is the three-dimensional visualization of macromolecular structures and molecular machines in their native hydrated state. Their physical fixation within ultra-thin vitrified ice layers is the crucial starting point.

scholarly journals Biological Applications at the Cutting Edge of Cryo-Electron Microscopy

2018 ◽  
Vol 24 (4) ◽  
pp. 406-419 ◽  
Author(s):  
Rebecca S. Dillard ◽  
Cheri M. Hampton ◽  
Joshua D. Strauss ◽  
Zunlong Ke ◽  
Deanna Altomara ◽  
...  
Keyword(s):  
Biological Sciences ◽  
Electron Microscopy ◽  
Sample Preparation ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Biological Applications ◽  
Preparation Methods ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Phase Plates

AbstractCryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

Simulation of three Dimensional Defect Images in Transmission Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 470-471
Author(s):  
W. D. Cooper ◽  
C. S. Hartley ◽  
J. J. Hren
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Three Dimensional ◽  
Crystalline Lattice ◽  
Continuum Models ◽  
Thin Foils ◽  
Transmission Electron ◽  
Microscope Images ◽  
General Computer Program ◽  
General Computer

Interpretation of electron microscope images of crystalline lattice defects can be greatly aided by computer simulation of theoretical contrast from continuum models of such defects in thin foils. Several computer programs exist at the present time, but none are sufficiently general to permit their use as an aid in the identification of the range of defect types encountered in electron microscopy. This paper presents progress in the development of a more general computer program for this purpose which eliminates a number of restrictions contained in other programs. In particular, the program permits a variety of foil geometries and defect types to be simulated.The conventional approximation of non-interacting columns is employed for evaluation of the two-beam dynamical scattering equations by a piecewise solution of the Howie-Whelan equations.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

The annealed (0001) α-alumina surface and its influence on thin-film growth

1995 ◽  
Vol 53 ◽  
pp. 336-337
Author(s):  
Michael W. Bench ◽  
Paul G. Kotula ◽  
C. Barry Carter
Keyword(s):  
Electron Microscopy ◽  
Surface Energy ◽  
Film Growth ◽  
Thin Film Growth ◽  
Energy Calculations ◽  
Transmission Electron ◽  
Reflection Electron Microscopy ◽  
Low Energy Electron ◽  
Surface Nature

The growth of semiconductors, superconductors, metals, and other insulators has been investigated using alumina substrates in a variety of orientations. The surface state of the alumina (for example surface reconstruction and step nature) can be expected to affect the growth nature and quality of the epilayers. As such, the surface nature has been studied using a number of techniques including low energy electron diffraction (LEED), reflection electron microscopy (REM), transmission electron microscopy (TEM), molecular dynamics computer simulations, and also by theoretical surface energy calculations. In the (0001) orientation, the bulk alumina lattice can be thought of as a layered structure with A1-A1-O stacking. This gives three possible terminations of the bulk alumina lattice, with theoretical surface energy calculations suggesting that termination should occur between the Al layers. Thus, the lattice often has been described as being made up of layers of (Al-O-Al) unit stacking sequences. There is a 180° rotation in the surface symmetry of successive layers and a total of six layers are required to form the alumina unit cell.

scholarly journals Structural Analysis of Jumbo Coliphage phAPEC6

2020 ◽  
Vol 21 (9) ◽  
pp. 3119 ◽  
Author(s):  
Jeroen Wagemans ◽  
Jessica Tsonos ◽  
Dominique Holtappels ◽  
Kiandro Fortuna ◽  
Jean-Pierre Hernalsteens ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Capsid Protein ◽  
Tem Analysis ◽  
Host Interaction ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Tail Sheath ◽  
Associated Proteins ◽  
Contractile Tail ◽  
Phage Capsid

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction.

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