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.

scholarly journals A simple pressure-assisted method for cryo-EM specimen preparation

2019 ◽  
Author(s):  
J. Zhao ◽  
H. Xu ◽  
M. Carroni ◽  
H. Lebrette ◽  
K. Wallden ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Pressure Gradient ◽  
Structural Biology ◽  
Specimen Preparation ◽  
Single Particles ◽  
Simple Method ◽  
Cryo Electron Microscopy ◽  
Low Concentrations ◽  
Wide Range ◽  
Major Bottleneck

AbstractCryo-electron microscopy (cryo-EM) has made great impacts on structural biology. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing cryo-EM specimens, named Preassis, in which the excess liquid is removed by introducing a pressure gradient through the EM grid. We show the unique advantages of Preassis in handling samples with low concentrations of protein single particles and micro-crystals in a wide range of buffer conditions.

scholarly journals Amorphous nickel titanium alloy film: a new choice for cryo electron microscopy sample preparation

2020 ◽  
Author(s):  
Xiaojun Huang ◽  
Lei Zhang ◽  
Zuoling Wen ◽  
Hui Chen ◽  
Shuoguo Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Titanium Alloy ◽  
Data Collection ◽  
Sample Preparation ◽  
Particle Distribution ◽  
Carbon Film ◽  
Nickel Titanium ◽  
Pure Gold ◽  
Interface Problem ◽  
Cryo Electron Microscopy

ABSTRACTCryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increase successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including superior electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

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.

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

scholarly journals A basic introduction to single particles cryo-electron microscopy

2021 ◽  
Vol 9 (1) ◽  
pp. 5-20
Author(s):  
Vittoria Raimondi ◽  
◽  
Alessandro Grinzato ◽  
◽  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Protein Complexes ◽  
Computational Power ◽  
Single Particles ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
First Choice ◽  
New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

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.

A New Method for Single Atom Specimen Preparation

1975 ◽  
Vol 33 ◽  
pp. 224-225
Author(s):  
W. Chiu ◽  
R. M. Glaeser ◽  
S. Prussin
Keyword(s):  
Unit Area ◽  
Carbon Film ◽  
Gold Atom ◽  
High Resolution Electron Microscopy ◽  
Carbon Films ◽  
Single Atom ◽  
Specimen Preparation ◽  
Electron Microscopic ◽  
Thin Carbon ◽  
Thin Carbon Film

A reliable method of confirming that one is able to produce single atom images in high resolution electron microscopy is to make a comparison of the number of atoms per unit area that is seen by the electron microscopic observations and the number per unit area as determined by a separate experimental technique of high accuracy. This report presents a method of preparinga specimen composed of a low distribution of gold atoms (ca. 1 atom/104 Å2) sandwiched between two ultra-thin carbon films, and also a method of measuring the gold atom density in the specimen by neutron activation analysis.The specimen was prepared by evaporation onto a freshly cleaved mica substrate in a vacuum evaporator, as shown in Figure 1. One side of the vacuum chamber was used to prepare a thin carbon film according to the method of Williams and Glaeser (1972), and the other side was used to evaporate the gold atoms from a molybdenum strip, which had a micro-quantity of gold deposited on a 1 cm2 surface area from a previous evaporation.

Electron Microscopic Comparison of Frozen-Hydrated with Negatively Stained and Rotary-Shadowed Creatine Kinase Single Molecules

1990 ◽  
Vol 48 (1) ◽  
pp. 262-263
Author(s):  
H. Winkler ◽  
T. Schnyder ◽  
U. Lücken
Keyword(s):  
Electron Microscopy ◽  
Creatine Kinase ◽  
Scanning Transmission Electron Microscopy ◽  
Carbon Films ◽  
Mass Measurements ◽  
Buffer Solution ◽  
Electron Microscopic ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Scanning Transmission

The mitochondrial isoform of creatine kinase isolated from chicken cardiac muscle consists predominantly of an octameric protein species with an Mr of 364000 and a small fraction of a dimeric species (Mr=86000). Electron micrographs of negatively stained creatine kinase show the square-shaped octameric molecules (Fig. 3) and the banana-shaped dimers. Mass measurements of individual molecules by scanning transmission electron microscopy yield about 340 kDa for the octamer, and 89 kDa for the dimer. After freeze-drying and high-resolution shadowing the surface of the octameric molecules shows a distinct cross-like division into four units (Fig. 4). The image analysis with circular harmonic averaging reveals a pronounced four-fold symmetry of both the rotary-shadowed and negatively stained octamers.Unstained creatine kinase molecules are investigated with cryo-electron microscopy. The samples were diluted with buffer solution to about 1 mg/ml. Drops of the suspension were applied to glow-discharged holey carbon films, blotted to remove excess liquid, immediately plunged into partially solidified liquid ethane, and stored under liquid nitrogen until insertion into the microscope. The specimens were examined in the DEEKO 250 electron microscope, operated at 100 kV. Micrographs were recorded by minimum-dose methods at a magnification of 60000 and an underfocus of about 1000 nm (Fig. 1).

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