scholarly journals Structure of the fission yeast actomyosin ring during constriction

2017 ◽  
Author(s):  
Matthew T. Swulius ◽  
Lam T. Nguyen ◽  
Mark S. Ladinsky ◽  
Davi R. Ortega ◽  
Samya Aich ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Actin Filaments ◽  
Focused Ion Beam ◽  
Nearest Neighbor ◽  
Ion Beam ◽  
Three Dimensional ◽  
Model Organism ◽  
Leading Edge ◽  
Specimen Preparation ◽  
Electron Cryotomography

AbstractCell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane remains unclear. Here we use cryosectioning and cryo-focused ion beam milling to gain access to cryo-preserved actomyosin rings in Schizosaccharomyces pombe for direct three-dimensional imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of approximately 150 nm in diameter that “saddles” the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane and angular distribution with respect to the membrane.Significance StatementMost eukaryotic cells divide using a contractile actomyosin ring, but its structure is unknown. Here we use new specimen preparation methods and electron cryotomography to image constricting rings directly in 3D, in a near-native state in the model organism Schizosaccharomyces pombe. Our images reveal the arrangement of individual actin filaments within the contracting actomyosin ring.

scholarly journals Structure of the fission yeast actomyosin ring during constriction

2018 ◽  
Vol 115 (7) ◽  
pp. E1455-E1464 ◽  
Author(s):  
Matthew T. Swulius ◽  
Lam T. Nguyen ◽  
Mark S. Ladinsky ◽  
Davi R. Ortega ◽  
Samya Aich ◽  
...  
Keyword(s):  
Angular Distribution ◽  
Actin Filaments ◽  
Direct Contact ◽  
3D Imaging ◽  
Nearest Neighbor ◽  
Ion Beam ◽  
Leading Edge ◽  
Electron Cryotomography ◽  
Nearest Neighbor Distances ◽  
Gain Access

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that “saddles” the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane.

scholarly journals Specimen preparation for three-dimensional atom probe using the focused ion-beam lift-out technique

2011 ◽  
Vol 17 (3) ◽  
pp. 292-295 ◽  
Author(s):  
T. Yamamoto ◽  
Y. Hanaoka ◽  
N. Mayama ◽  
T. Kaito ◽  
T. Adachi ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Atom Probe ◽  
Specimen Preparation

scholarly journals Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Benjamin D Engel ◽  
Miroslava Schaffer ◽  
Luis Kuhn Cuellar ◽  
Elizabeth Villa ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Carbon Fixation ◽  
Nearest Neighbor ◽  
Electron Tomography ◽  
Close Association ◽  
Ion Beam ◽  
Three Dimensional ◽  
Chloroplast Envelope ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

Chloroplast function is orchestrated by the organelle's intricate architecture. By combining cryo-focused ion beam milling of vitreous Chlamydomonas cells with cryo-electron tomography, we acquired three-dimensional structures of the chloroplast in its native state within the cell. Chloroplast envelope inner membrane invaginations were frequently found in close association with thylakoid tips, and the tips of multiple thylakoid stacks converged at dynamic sites on the chloroplast envelope, implicating lipid transport in thylakoid biogenesis. Subtomogram averaging and nearest neighbor analysis revealed that RuBisCO complexes were hexagonally packed within the pyrenoid, with ∼15 nm between their centers. Thylakoid stacks and the pyrenoid were connected by cylindrical pyrenoid tubules, physically bridging the sites of light-dependent photosynthesis and light-independent carbon fixation. Multiple parallel minitubules were bundled within each pyrenoid tubule, possibly serving as conduits for the targeted one-dimensional diffusion of small molecules such as ATP and sugars between the chloroplast stroma and the pyrenoid matrix.

scholarly journals A FIB Micro-Sampling Technique for Three-Dimensional Characterization of a Site-Specific Defect

2004 ◽  
Vol 12 (6) ◽  
pp. 26-29 ◽  
Author(s):  
Toshie Yaguchi ◽  
Yasushi Kuroda ◽  
Mitsuru Konno ◽  
Takeo Kamino ◽  
Tsuyohsi Ohnishi ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Semiconductor Devices ◽  
Three Dimensional ◽  
Sampling Technique ◽  
Scanning Transmission Electron Microscope ◽  
Specimen Preparation ◽  
Specimen Holder ◽  
Scanning Transmission ◽  
A Site

In characterization or failure analysis of new materials and semiconductor devices, the requirements for three dimensional observation and analysis are rapidly increasing. We discuss techniques for specimen preparation, three-dimensional observation, and elemental analysis of semiconductor devices that we developed using a system consisting of a dedicated focused ion beam (FIB) instrument and a scanning transmission electron microscope (STEM). The system utilizes a FIB-STEM compatible specimen holder with a specially designed rotation mechanism, which allows 360° rotation of a specimen on a conical stage (needle stub) around the ion beam axis of the FIB system and 360° rotation perpendicular to the electron beam in the STEM. A piece of sample (micro sample) is extracted from a specific-site by the micro-sampling technique and mounted on the needle stub. Instruments used in the study are the Hitachi FB-2100 FIB system with a micro-sampling attachment and the HD-2300 field emission 200kV STEM.

scholarly journals Molecular plasticity of the native mouse skeletal sarcomere revealed by cryo-ET

2020 ◽  
Author(s):  
Zhexin Wang ◽  
Michael Grange ◽  
Thorsten Wagner ◽  
Ay Lin Kho ◽  
Mathias Gautel ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Volume Averaging ◽  
Molecular Architecture ◽  
Molecular Detail ◽  
Cross Links ◽  
Molecular Plasticity

AbstractSarcomeres are the force-generating and load-bearing devices of muscles. A precise molecular understanding of how the entire sarcomere is built is required to understand its role in health, disease and ageing. Here, we determine the in situ molecular architecture of vertebrate skeletal sarcomeres through electron cryo-tomography of cryo-focused ion beam-milled native myofibrils. The reconstructions reveal the three-dimensional organisation and interaction of actin and myosin filaments in the A-band, I-band and Z-disc and demonstrate how α -actinin cross-links antiparallel actin filaments to form a mesh-like structure in the Z-disc at an unprecedented level of molecular detail. A prominent feature is a so-far undescribed doublet of α-actinin cross-links with ∼ 6 nm spacing. Sub-volume averaging shows the interaction between myosin, tropomyosin and actin in molecular detail at ∼ 10 Å resolution and reveals two coexisting conformations of actin-bound heads. The flexible orientation of the lever arm and the essential and regulatory light chains allow the two heads of the “double-headed” myosin not only to interact with the same actin filament but also to split between two actin filaments. Our results provide new insights into the conformational plasticity and fundamental organisation of vertebrate skeletal muscle and serve as a strong foundation for future in situ investigations of muscle diseases.

Overview: Recent Progress in Three-Dimensional Atom Probe Instruments and Applications

2007 ◽  
Vol 13 (6) ◽  
pp. 408-417 ◽  
Author(s):  
Alfred Cerezo ◽  
Peter H. Clifton ◽  
Sergio Lozano-Perez ◽  
Peter Panayi ◽  
Gang Sha ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Materials Science ◽  
Ion Beam ◽  
Three Dimensional ◽  
Atom Probe ◽  
High Mass ◽  
Large Field ◽  
Specimen Preparation ◽  
Preparation Methods ◽  
Energy Compensation

Over the last few years there have been significant developments in the field of three-dimensional atom probe (3DAP) analysis. This article reviews some of the technical compromises that have led to different instrument designs and the recent improvements in performance. An instrument has now been developed, based around a novel reflectron configuration combining both energy compensation and focusing elements, that yields a large field of view and very high mass resolution. The use of laser pulsing in the 3DAP, together with developments in specimen preparation methods using a focused ion-beam instrument, have led to a significant widening in the range of materials science problems that can be addressed with the 3DAP. Recent studies of semiconductor materials and devices are described.

Low Energy Ar Ion Milling of FIB TEM Specimens from 14 nm and Future FinFET Technologies

2018 ◽  
Author(s):  
C.S. Bonifacio ◽  
P. Nowakowski ◽  
M.J. Campin ◽  
M.L. Ray ◽  
P.E. Fischione
Keyword(s):  
Field Effect Transistor ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Specimen Preparation ◽  
Ion Milling ◽  
Transmission Electron ◽  
High Resolution Tem ◽  
Effect Transistor ◽  
20 Nm ◽  
Argon Ion

Abstract Transmission electron microscopy (TEM) specimens are typically prepared using the focused ion beam (FIB) due to its site specificity, and fast and accurate thinning capabilities. However, TEM and high-resolution TEM (HRTEM) analysis may be limited due to the resulting FIB-induced artifacts. This work identifies FIB artifacts and presents the use of argon ion milling for the removal of FIB-induced damage for reproducible TEM specimen preparation of current and future fin field effect transistor (FinFET) technologies. Subsequently, high-quality and electron-transparent TEM specimens of less than 20 nm are obtained.

Focused Ion Beam Analysis of Low-K Dielectrics

2000 ◽  
Author(s):  
H. J. Bender ◽  
R. A. Donaton
Keyword(s):  
Electron Microscope ◽  
Cross Section ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Single Step ◽  
Specimen Preparation ◽  
Transmission Electron ◽  
Beam Analysis ◽  
Low K ◽  
Scanning Electron

Abstract The characteristics of an organic low-k dielectric during investigation by focused ion beam (FIB) are discussed for the different FIB application modes: cross-section imaging, specimen preparation for transmission electron microscopy, and via milling for device modification. It is shown that the material is more stable under the ion beam than under the electron beam in the scanning electron microscope (SEM) or in the transmission electron microscope (TEM). The milling of the material by H2O vapor assistance is strongly enhanced. Also by applying XeF2 etching an enhanced milling rate can be obtained so that both the polymer layer and the intermediate oxides can be etched in a single step.

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.

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