A Fully Automated System for the Preparation of Samples for Cryo-Electron Microscopy

2010 ◽  
Author(s):  
Zachary J. Thompson ◽  
Kevin L. Johnson ◽  
Nicolas Overby ◽  
Jessica I. Chidi ◽  
William K. Pryor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Environmental Control ◽  
Human Interaction ◽  
Automated System ◽  
Specimen Preparation ◽  
Cryo Electron Microscopy ◽  
Early Testing ◽  
High Humidity Environment ◽  
Fully Automated System

The preparation of specimens for cryo-electron microscopy is currently a labor and time intensive process, and the quality of resulting samples is highly dependent on both environmental and procedural factors. Specimens must be applied to sample grids in a high-humidity environment, frozen in liquid ethane, and stored in liquid nitrogen. The combination of cryogenic temperatures and humidity-control mandates the segregation of the humidity-controlled environment from the cryogenic environment. Several devices which automate portions of the specimen preparation process are currently in use; however, these systems still require significant human interaction in order to create viable samples. This paper describes a fully automated system for specimen preparation. The resulting system removes the need for human input during specimen preparation, improves process control, and provides similar levels of environmental control. Early testing shows that the resulting system is capable of manipulating samples in an autonomous manner while providing performance similar to existing systems.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

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 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

2020 ◽  
Vol 212 (3) ◽  
pp. 107633 ◽  
Author(s):  
Florian Fäßler ◽  
Bettina Zens ◽  
Robert Hauschild ◽  
Florian K.M. Schur
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Specimen Preparation ◽  
Cryo Electron Microscopy ◽  
3D Printed

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

scholarly journals GPU-accelerated analysis and visualization of large structures solved by molecular dynamics flexible fitting

2014 ◽  
Vol 169 ◽  
pp. 265-283 ◽  
Author(s):  
John E. Stone ◽  
Ryan McGreevy ◽  
Barry Isralewitz ◽  
Klaus Schulten
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Hybrid Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Interactive Computation ◽  
Biomolecular Complexes ◽  
Quality Of Fit

Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithmsvis-à-visexisting tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.

Wide Field Electron Microscopy: The Sublimation of Epon-Araldite

1973 ◽  
Vol 31 ◽  
pp. 366-367
Author(s):  
Grace C. H. Yang ◽  
A. B. Morrison
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Specimen Preparation ◽  
Wide Field ◽  
Electron Micrograph ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Scanning Range

To emphasize the best feature of this technique, the term “wide field” is used throughout the text instead of the usual “scanning range magnification” or “low magnification”. Wide field electron microscopy presently is not being fully utilized in many laboratories, perhaps because the quality of the electron micrograph obtained is not worthy of the supposedly long and tedious specimen preparation. We report here that the quality of the wide field electron micrograph can be greatly enhanced by using the old “sublimation trick” for the methacrylate section.Two kinds of embedment formula were tested: Luft's Epon (mixture A:B= 6;4), and Mollenhauer's Epon-Araldite mixture 1. Only the Epon-Araldite showed the “foot print” of the electron beam (fig. 1).

scholarly journals Experimental Characterization and Mitigation of Specimen Charging on Thin Films with One Conducting Layer

2004 ◽  
Vol 10 (6) ◽  
pp. 783-789 ◽  
Author(s):  
Kenneth H. Downing ◽  
M.R. McCartney ◽  
Robert M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Data Collection ◽  
High Variability ◽  
Conducting Layer ◽  
Experimental Characterization ◽  
Cryo Electron Microscopy ◽  
Series Of Experiments ◽  
Significant Factors

Specimen charging may be one of the most significant factors that contribute to the high variability and generally low quality of images in cryo-electron microscopy. Understanding the nature of specimen charging can help in devising methods to reduce or even avoid its effects and thus improve the rate of data collection as well as the quality of the data. We describe a series of experiments that help to characterize the charging phenomenon, which has been termed the Berriman effect. The pattern of buildup and disappearance of the charge pattern has led to several suggestions for how to alleviate the effect. Experiments are described that demonstrate the feasibility of such charge mitigation.

An Improved Holey Carbon Film for Cryo-Electron Microscopy

2007 ◽  
Vol 13 (5) ◽  
pp. 365-371 ◽  
Author(s):  
Joel Quispe ◽  
John Damiano ◽  
Stephen E. Mick ◽  
David P. Nackashi ◽  
Denis Fellmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Particle Distribution ◽  
Carbon Film ◽  
Soft Materials ◽  
Carbon Films ◽  
Water Soluble ◽  
Specimen Preparation ◽  
Dramatic Improvement ◽  
Single Particles ◽  
Cryo Electron Microscopy

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

2004 ◽  
Vol 146 (3) ◽  
pp. 325-333 ◽  
Author(s):  
Nobuhiko Gyobu ◽  
Kazutoshi Tani ◽  
Yoko Hiroaki ◽  
Akiko Kamegawa ◽  
Kaoru Mitsuoka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Specimen Preparation ◽  
Sandwich Technique ◽  
Cryo Electron Microscopy
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