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 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.

scholarly journals Introduction to high-resolution cryo-electron microscopy

2016 ◽  
Vol 62 (3) ◽  
pp. 383-394
Author(s):  
Mariusz Czarnocki-Cieciura ◽  
Marcin Nowotny
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes

For many years two techniques have dominated structural biology – X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6–10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the “resolution revolution” in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

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.

scholarly journals The dynamic chirality flips of Skyrmion bubbles

2021 ◽  
Author(s):  
Yuan Yao ◽  
Bei Ding ◽  
Jinjing Liang ◽  
Hang Li ◽  
Xi Shen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Magnetic Domain ◽  
Two Dimensions ◽  
Entire Space ◽  
Transmission Electron ◽  
Fundamental Research ◽  
Lorentz Transmission Electron Microscopy ◽  
Spin Texture ◽  
Magnetic Vectors

Abstract Magnetic skyrmion, a topological magnetic domain with complex non-coplanar spin texture, appears a disk-like structure in two dimensions. Exploring three-dimensional spin texture and related chirality switching has drawn enormous interests from the perspective of fundamental research. Here, the three-dimensional magnetic moment of the skyrmion bubbles in centrosymmetric Mn-Ni-Ga were reconstructed with the vector field tomography approach via Lorentz transmission electron microscopy. The type of the bubbles was determined from investigating the magnetic vectors in entire space. We found that the bubbles switched their chirality easily but still keep the polarity to remain the singularity of the bubbles within the material. Our results offer valuable insights into the fundamental mechanisms underlying the spin chirality flips dynamics of skyrmion bubbles.

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.

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.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

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.

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