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

2020 ◽  
Author(s):  
Florian Fäßler ◽  
Bettina Zens ◽  
Robert Hauschild ◽  
Florian K.M. Schur
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Electron Tomography ◽  
Low Cost ◽  
Ion Beam ◽  
Control Cell ◽  
Specimen Preparation ◽  
Micro Patterning ◽  
Cryo Electron Microscopy ◽  
3D Printed

AbstractCryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.

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 Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

2019 ◽  
Author(s):  
Mauricio Toro-Nahuelpan ◽  
Ievgeniia Zagoriy ◽  
Fabrice Senger ◽  
Laurent Blanchoin ◽  
Manuel Théry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Specimen Preparation ◽  
Cell Architecture ◽  
Molecular Machinery ◽  
Cell Positioning ◽  
Related Proteins

AbstractSpatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex patterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell 3D-structural characterization of the underlying molecular machinery.

scholarly journals Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

2019 ◽  
Author(s):  
Mauricio Toro-Nahuelpan ◽  
Ievgeniia Zagoriy ◽  
Fabrice Senger ◽  
Laurent Blanchoin ◽  
Manuel Théry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Carbon Films ◽  
Specimen Preparation ◽  
Complex Shapes ◽  
Cell Positioning ◽  
Related Proteins

Abstract Micromachining by cryo-focused ion beam (FIB) milling coupled to cryo-electron tomography (ET) enables visualization of macromolecules directly inside cells. Yet, spatial control of cell adhesion on electron microscopy (EM) grids remains a bottleneck in the specimen preparation pipeline. This protocol describes a contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We achieved accurate and reproducible cell positioning, leading to optimized preparations for cryo-FIB milling. We tested HeLa and RPE1 cell lines on various grid types (gold or titanium mesh coated with SiO2, gold or carbon films). Briefly, grids were passivated with an anti-fouling agent, followed by controlled ablation of the passivation layer, and further functionalization with fibronectin. The micropatterning procedure takes ~3 h. Employing micropatterning to produce complex shapes generated a predictable intracellular organization, allowing direct correlation between cellular architecture and in-cell 3D-structural characterization of the underlying machinery at molecular resolution.

Transmission Electron Microscopy (TEM) Specimen Preparation Technique using Focused Ion Beam (FIB): Application to Material Characterization of Chemical Vapor Deposition of Tungsten (W) and Tungsten Silicides (WSix)

1997 ◽  
Author(s):  
K. Doong ◽  
J.-M. Fu ◽  
Y.-C. Huang
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Chemical Vapor Deposition ◽  
Vapor Deposition ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Chemical Vapor ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Transmission Electron

Abstract The specimen preparation technique using focused ion beam (FIB) to generate cross-sectional transmission electron microscopy (XTEM) samples of chemical vapor deposition (CVD) of Tungsten-plug (W-plug) and Tungsten Silicides (WSix) was studied. Using the combination method including two axes tilting[l], gas enhanced focused ion beam milling[2] and sacrificial metal coating on both sides of electron transmission membrane[3], it was possible to prepare a sample with minimal thickness (less than 1000 A) to get high spatial resolution in TEM observation. Based on this novel thinning technique, some applications such as XTEM observation of W-plug with different aspect ratio (I - 6), and the grain structure of CVD W-plug and CVD WSix were done. Also the problems and artifacts of XTEM sample preparation of high Z-factor material such as CVD W-plug and CVD WSix were given and the ways to avoid or minimize them were suggested.

A Methodology to Reduce Ion Beam Induced Damage in TEM Specimens Prepared by FIB

2002 ◽  
Author(s):  
Chin Kai Liu ◽  
Chi Jen. Chen ◽  
Jeh Yan.Chiou ◽  
David Su
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Sample Preparation ◽  
Integrated Circuit ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Specimen Preparation ◽  
Tem Analysis ◽  
Sensitive Issue ◽  
Transmission Electron

Abstract Focused ion beam (FIB) has become a useful tool in the Integrated Circuit (IC) industry, It is playing an important role in Failure Analysis (FA), circuit repair and Transmission Electron Microscopy (TEM) specimen preparation. In particular, preparation of TEM samples using FIB has become popular within the last ten years [1]; the progress in this field is well documented. Given the usefulness of FIB, “Artifact” however is a very sensitive issue in TEM inspections. The ability to identify those artifacts in TEM analysis is an important as to understanding the significance of pictures In this paper, we will describe how to measure the damages introduced by FIB sample preparation and introduce a better way to prevent such kind of artifacts.

scholarly journals Gold, Silver, and Electrum Electroless Plating on Additively Manufactured Laser Powder-Bed Fusion AlSi10Mg Parts: A Review

Coatings ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 422
Author(s):  
Dana Ashkenazi ◽  
Alexandra Inberg ◽  
Yosi Shacham-Diamand ◽  
Adin Stern
Keyword(s):  
Additive Manufacturing ◽  
Low Cost ◽  
Ion Beam ◽  
Surface Finishing ◽  
Powder Bed Fusion ◽  
Laser Powder Bed Fusion ◽  
Powder Bed ◽  
Finishing Process ◽  
Gold Silver ◽  
3D Printed

Additive manufacturing (AM) revolutionary technologies open new opportunities and challenges. They allow low-cost manufacturing of parts with complex geometries and short time-to-market of products that can be exclusively customized. Additive manufactured parts often need post-printing surface modification. This study aims to review novel environmental-friendly surface finishing process of 3D-printed AlSi10Mg parts by electroless deposition of gold, silver, and gold–silver alloy (e.g., electrum) and to propose a full process methodology suitable for effective metallization. This deposition technique is simple and low cost method, allowing the metallization of both conductive and insulating materials. The AlSi10Mg parts were produced by the additive manufacturing laser powder bed fusion (AM-LPBF) process. Gold, silver, and their alloys were chosen as coatings due to their esthetic appearance, good corrosion resistance, and excellent electrical and thermal conductivity. The metals were deposited on 3D-printed disk-shaped specimens at 80 and 90 °C using a dedicated surface activation method where special functionalization of the printed AlSi10Mg was performed to assure a uniform catalytic surface yielding a good adhesion of the deposited metal to the substrate. Various methods were used to examine the coating quality, including light microscopy, optical profilometry, XRD, X-ray fluorescence, SEM–energy-dispersive spectroscopy (EDS), focused ion beam (FIB)-SEM, and XPS analyses. The results indicate that the developed coatings yield satisfactory quality, and the suggested surface finishing process can be used for many AM products and applications.

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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