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.

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.

Tailoring surface acoustic wave atomisation for cryo-electron microscopy sample preparation

Lab on a Chip ◽  
2019 ◽  
Vol 19 (8) ◽  
pp. 1378-1385 ◽  
Author(s):  
Dariush Ashtiani ◽  
Alex de Marco ◽  
Adrian Neild
Keyword(s):  
Electron Microscopy ◽  
Fluid Flow ◽  
Sample Preparation ◽  
Acoustic Wave ◽  
Surface Acoustic Wave ◽  
Flow Rate ◽  
Fluid Flow Rate ◽  
Cryo Electron Microscopy ◽  
Electron Microscopy Grid

Surface acoustic wave (SAW) atomisation is investigated in the context of cryo electron microscopy grid preparation. Here, the primary requirements are a reproducible and narrow plume of droplets delivering a low fluid flow rate.

scholarly journals Improving the Resolution of Sputter-coated Films

2000 ◽  
Vol 8 (2) ◽  
pp. 16-17
Author(s):  
Mary Mager
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Fine Structure ◽  
High Resolution ◽  
Carbon Film ◽  
Metal Films ◽  
Pure Gold ◽  
High Magnification ◽  
Conducting Film ◽  
Scanning Electron

After an inquiry from the Microscopy Listserver, I went back to my 1980 copy of Scanning Electron Microscopy, volume I. Several authors had investigated the structure of thin metal films by depositing the films onto carbon-film-covered TEM grids and imaging the films at high magnification. There were several proposals for new devices that have since become standards for high-resolution coaters, but the Listserver inquiry was for a fine conducting film suitabie for high-resolution SEM from an existing sputter coater.There were several factors studied that influenced the fine structure of the films. The first was the materials sputtered: for a given set of conditions of voltage, current and time, platinum gave the finest film, 60% gold-40% palladium (Au/Pd) the next finest and pure gold the least fine.

scholarly journals Antibody-Based Affinity Cryo-Electron Microscopy at 2.6 Å Resolution

2016 ◽  
Author(s):  
Guimei Yu ◽  
Kunpeng Li ◽  
Pengwei Huang ◽  
Xi Jiang ◽  
Wen Jiang
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
High Concentration ◽  
Low Concentration ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Grid Approach ◽  
Reconstruction Quality ◽  
Tulane Virus ◽  
Quantitative Analyses

AbstractThe affinity cryo-electron microscopy (cryo-EM) approach has been explored in recent years to simplify and improve the sample preparation for cryo-EM. Despite the demonstrated successes for low-concentration and unpurified specimens, the lack of near-atomic structures using this approach has led to a common perception of affinity cryo-EM as a niche technique incapable of reaching high resolutions. Here, we report a ~2.6 Å structure solved using the antibody-based affinity grid approach with a Tulane virus sample of low concentration. This is the first near-atomic structure solved using the affinity cryo-EM approach. Quantitative analyses of the structure indicate data and reconstruction quality comparable to conventional grid preparation method using samples at high concentration. With the shifting of bottlenecks of cryo-EM structural studies to sample grid preparation, our demonstration of the sub-3 Å capability of affinity cryo-EM approach indicates its potential in revolutionizing cryo-EM sample preparation for a broader spectrum of specimens.

scholarly journals Real-time object locator for cryo-EM data collection --- You only navigate EM once ---

2021 ◽  
Author(s):  
Koji Yonekura ◽  
Saori Maki-Yonekura ◽  
Hisashi Naitow ◽  
Tasuku Hamaguchi ◽  
Kiyofumi Takaba
Keyword(s):  
Electron Microscopy ◽  
Machine Learning ◽  
Electron Diffraction ◽  
Data Collection ◽  
Real Time ◽  
Single Particle ◽  
Detection System ◽  
Target Object ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy

In cryo-electron microscopy (cryo-EM) data collection, locating a target object is the most error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation showed its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and for locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection.

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