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 Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

2019 ◽  
Author(s):  
Andrea Fera ◽  
Qianping He ◽  
Guofeng Zhang ◽  
Richard D. Leapman
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Blood Platelets ◽  
Ion Beam ◽  
Simple Expression ◽  
Heavy Atoms ◽  
3D Electron Microscopy ◽  
Block Face ◽  
Microscopy Techniques

SummaryStain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.

Method for Cross-sectional Thin Specimen Preparation from a Specific Site Using a Combination of a Focused Ion Beam System and Intermediate Voltage Electron Microscope and Its Application to the Characterization of a Precipitate in a Steel

2001 ◽  
Vol 7 (3) ◽  
pp. 287-291
Author(s):  
Toshie Yaguchi ◽  
Hiroaki Matsumoto ◽  
Takeo Kamino ◽  
Tohru Ishitani ◽  
Ryoichi Urao
Keyword(s):  
Electron Microscope ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Specific Site ◽  
Specimen Preparation ◽  
Thin Specimen ◽  
Cross Sectional ◽  
Electron Detector ◽  
Specimen Holder

AbstractIn this study, we discuss a method for cross-sectional thin specimen preparation from a specific site using a combination of a focused ion beam (FIB) system and an intermediate voltage transmission electron microscope (TEM). A FIB-TEM compatible specimen holder was newly developed for the method. The thinning of the specimen using the FIB system and the observation of inside structure of the ion milled area in a TEM to localize a specific site were alternately carried out. The TEM fitted with both scanning transmitted electron detector and secondary electron detector enabled us to localize the specific site in a halfway milled specimen with the positional accuracy of better than 0.1 µm. The method was applied to the characterization of a precipitate in a steel. A submicron large precipitate was thinned exactly at its center for the characterization by a high-resolution electron microscopy and an elemental mapping.

Characterization of FIB Damage in Silicon

1999 ◽  
Vol 5 (S2) ◽  
pp. 740-741 ◽  
Author(s):  
C.A. Urbanik ◽  
B.I. Prenitzer ◽  
L.A. Gianhuzzi ◽  
S.R. Brown ◽  
T.L. Shofner ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Target Material ◽  
Beam Current ◽  
Specimen Preparation ◽  
Transfer Energy ◽  
Transmission Electron ◽  
Preparation Techniques ◽  
Reactive Gas

Focused ion beam (FIB) instruments are useful for high spatial resolution milling, deposition, and imaging capabilities. As a result, FIB specimen preparation techniques have been widely accepted within the semiconductor community as a means to rapidly prepare high quality, site-specific specimens for transmission electron microscopy (TEM) [1]. In spite of the excellent results that have been observed for both high resolution (HREM) and standard TEM specimen preparation applications, a degree of structural modification is inherent to FIB milled surfaces [2,3]. The magnitude of the damage region that results from Ga+ ion bombardment is dependent on the operating parameters of the FIB (e.g., beam current, beam voltage, milling time, and the use of reactive gas assisted etching).Lattice defects occur as a consequence of FIB milling because the incident ions transfer energy to the atoms of the target material. Momentum transferred from the incident ions to the target atoms can result in the creation of point defects (e.g., vacancies, self interstitials, and interstitial and substitutional ion implantation), the generation of phonons, and plasmon excitation in the case of metal targets.

scholarly journals Fabrication and Characterization of Solid Composite Yarns from Carbon Nanotubes and Poly(dicyclopentadiene)

Nanomaterials ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 717 ◽  
Author(s):  
Wenbo Xin ◽  
Joseph Severino ◽  
Arie Venkert ◽  
Hang Yu ◽  
Daniel Knorr ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Carbon Nanotubes ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Strengthening Mechanism ◽  
Composite Yarn ◽  
Transmission Electron ◽  
Frictional Forces ◽  
Mechanical Consolidation

In this report, networks of carbon nanotubes (CNTs) are transformed into composite yarns by infusion, mechanical consolidation and polymerization of dicyclopentadiene (DCPD). The microstructures of the CNT yarn and its composite are characterized by scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), and a focused ion beam used for cross-sectioning. Pristine yarns have tensile strength, modulus and elongation at failure of 0.8 GPa, 14 GPa and 14.0%, respectively. In the composite yarn, these values are significantly enhanced to 1.2 GPa, 68 GPa and 3.4%, respectively. Owing to the consolidation and alignment improvement, its electrical conductivity was increased from 1.0 × 105 S/m (raw yarn) to 5.0 × 105 S/m and 5.3 × 105 S/m for twisted yarn and composite yarn, respectively. The strengthening mechanism is attributed to the binding of the DCPD polymer, which acts as a capstan and increases frictional forces within the nanotube bundles, making it more difficult to pull them apart.

Pre-Meeting Topical Conference

2002 ◽  
Vol 8 (I1) ◽  
pp. 20-20
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Low Voltage ◽  
Ion Beam ◽  
Analytical Electron Microscopy ◽  
Polymeric Materials ◽  
Specimen Preparation ◽  
Microbeam Analysis ◽  
Scanning Electron

Topic: Characterization of Non-Conductive or Charging Materials by Microbeam AnalysisThe goal of this topical conference is to present the state of the art for materials characterization of non-conductive or charging materials using microbeam analysis. Examples of charging materials include polymeric materials, ceramic materials, and photoresist materials in the microelectronic industry. Also, the characterization of biological specimens will be covered because they are prone to problems related to charging. These materials are of great technological importance and their characterization is still a great challenge because they charge when analyzed with an electron beam. The techniques of microbeam analysis that will be considered are: X-ray Microanalysis in the Electron Microprobe, Low Voltage Scanning Electron Microscopy, Environmental Scanning Electron Microscopy, Analytical Electron Microscopy with Field Emission Transmission Electron Microscopy, and Focused Ion Beam Milling for specimen preparation. World experts will present papers on these topics. Papers from this topical conference will be published in a special issue of Microscopy & Microanalysis.

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