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.

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 Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

2022 ◽  
Author(s):  
Jie E Yang ◽  
Matthew R Larson ◽  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Daniel Parrell ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Low Dose ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

scholarly journals Tipping solutions: emerging 3D nano-fabrication/ -imaging technologies

Nanophotonics ◽  
2017 ◽  
Vol 6 (5) ◽  
pp. 923-941 ◽  
Author(s):  
Gediminas Seniutinas ◽  
Armandas Balčytis ◽  
Ignas Reklaitis ◽  
Feng Chen ◽  
Jeffrey Davis ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Imaging Technique ◽  
Ion Beam ◽  
Three Dimensional ◽  
Molecular Systems ◽  
Force Microscopy ◽  
Nano Fabrication ◽  
Atomic Force ◽  
Scanning Electron

AbstractThe evolution of optical microscopy from an imaging technique into a tool for materials modification and fabrication is now being repeated with other characterization techniques, including scanning electron microscopy (SEM), focused ion beam (FIB) milling/imaging, and atomic force microscopy (AFM). Fabrication and in situ imaging of materials undergoing a three-dimensional (3D) nano-structuring within a 1−100 nm resolution window is required for future manufacturing of devices. This level of precision is critically in enabling the cross-over between different device platforms (e.g. from electronics to micro-/nano-fluidics and/or photonics) within future devices that will be interfacing with biological and molecular systems in a 3D fashion. Prospective trends in electron, ion, and nano-tip based fabrication techniques are presented.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

3D Observation of Elemental Distribution of Si-Device using a Dedicated FIB/STEM System

2005 ◽  
Author(s):  
T. Yaguchi ◽  
M. Konno ◽  
T. Kamino ◽  
M. Ogasawara ◽  
K. Kaji ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Elemental Distribution ◽  
Multivariate Statistical ◽  
X Ray ◽  
Sample Rotation ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Elemental Maps

Abstract A technique for preparation of a pillar shaped sample and its multi-directional observation of the sample using a focused ion beam (FIB) / scanning transmission electron microscopy (STEM) system has been developed. The system employs an FIB/STEM compatible sample rotation holder with a specially designed rotation mechanism, which allows the sample to be rotated 360 degrees [1-3]. This technique was used for the three dimensional (3D) elemental mapping of a contact plug of a Si device in 90 nm technology. A specimen containing a contact plug was shaped to a pillar sample with a cross section of 200 nm x 200 nm and a 5 um length. Elemental analysis was performed with a 200 kV HD-2300 STEM equipped with the EDAX genesis Energy dispersive X-ray spectroscopy (EDX) system. Spectrum imaging combined with multivariate statistical analysis (MSA) [4, 5] was used to enhance the weak X-ray signals of the doped area, which contain a low concentration of As-K. The distributions of elements, especially the dopant As, were successfully enhanced by MSA. The elemental maps were .. reconstructed from the maps.

Investigations of Leakage Paths in Sub-0.35μm DRAM Products Using Advanced Focused Ion Beam Techniques

1998 ◽  
Author(s):  
H. Lorenz ◽  
C. Engel
Keyword(s):  
Focused Ion Beam ◽  
Cell Function ◽  
Dielectric Breakdown ◽  
Ion Beam ◽  
Electrical Characterization ◽  
Floating Gate ◽  
Leakage Path ◽  
Contrast Technique ◽  
Voltage Contrast

Abstract Due to the continuously decreasing cell size of DRAMs and concomitantly diminishing thickness of some insulating layers new failure mechanisms appear which until now had no significance for the cell function. For example high resistance leakage paths between closely spaced conductors can lead to retention problems. These are hard to detect by electrical characterization in a memory tester because the involved currents are in the range of pA. To analyze these failures we exploit the very sensitive passive voltage contrast of the Focused Ion Beam Microscope (FIB). The voltage contrast can further be enhanced by in-situ FIB preparations to obtain detailed information about the failure mechanism. The first part of this paper describes a method to detect a leakage path between a borderless contact on n-diffusion and an adjacent floating gate by passive voltage contrast achieved after FIB circuit modification. In the second part we will demonstrate the localization of a DRAM trench dielectric breakdown. In this case the FIB passive voltage contrast technique is not limited to the localization of the failing trench. We can also obtain the depth of the leakage path by selective insitu etching with XeF2 stopped immediately after a voltage contrast change.

scholarly journals Investigation of the selective color-changing mechanism of Dynastes tityus beetle (Coleoptera: Scarabaeidae)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiyu Sun ◽  
Wei Wu ◽  
Limei Tian ◽  
Wei Li ◽  
Fang Zhang ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Color Change ◽  
Incidence Angle ◽  
Ion Beam ◽  
Three Dimensional ◽  
Water Infiltration ◽  
Photonic Materials ◽  
Local Color ◽  
Water Contact ◽  
Color Changes

AbstractNot only does the Dynastes tityus beetle display a reversible color change controlled by differences in humidity, but also, the elytron scale can change color from yellow-green to deep-brown in specified shapes. The results obtained by focused ion beam-scanning electron microscopy (FIB-SEM), show that the epicuticle (EPI) is a permeable layer, and the exocuticle (EXO) is a three-dimensional photonic crystal. To investigate the mechanism of the reversible color change, experiments were conducted to determine the water contact angle, surface chemical composition, and optical reflectance, and the reflective spectrum was simulated. The water on the surface began to permeate into the elytron via the surface elemental composition and channels in the EPI. A structural unit (SU) in the EXO allows local color changes in varied shapes. The reflectance of both yellow-green and deep-brown elytra increases as the incidence angle increases from 0° to 60°. The microstructure and changes in the refractive index are the main factors that influence the process of reversible color change. According to the simulation, the lower reflectance causing the color change to deep-brown results from water infiltration, which increases light absorption. Meanwhile, the waxy layer has no effect on the reflection of light. This study lays the foundation to manufacture engineered photonic materials that undergo controllable changes in iridescent color.

scholarly journals Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexey A. Polilov ◽  
Anastasia A. Makarova ◽  
Song Pang ◽  
C. Shan Xu ◽  
Harald Hess
Keyword(s):  
Electron Microscopy ◽  
Biological Samples ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Entire Volume ◽  
Simple Protocol ◽  
3D Electron Microscopy ◽  
3D Em

AbstractModern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

scholarly journals Cyclic Deformation of Microcantilevers Using In-Situ Micromanipulation

2021 ◽  
Author(s):  
A. H. S. Iyer ◽  
M. H. Colliander
Keyword(s):  
Degrees Of Freedom ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Structural Components ◽  
Cyclic Testing ◽  
Phase Boundaries ◽  
Bulk Specimen ◽  
Arbitrary Position ◽  
Three Degrees Of Freedom

Abstract Background The trend in miniaturisation of structural components and continuous development of more advanced crystal plasticity models point towards the need for understanding cyclic properties of engineering materials at the microscale. Though the technology of focused ion beam milling enables the preparation of micron-sized samples for mechanical testing using nanoindenters, much of the focus has been on monotonic testing since the limited 1D motion of nanoindenters imposes restrictions on both sample preparation and cyclic testing. Objective/Methods In this work, we present an approach for cyclic microcantilever bending using a micromanipulator setup having three degrees of freedom, thereby offering more flexibility. Results The method has been demonstrated and validated by cyclic bending of Alloy 718plus microcantilevers prepared on a bulk specimen. The experiments reveal that this method is reliable and produces results that are comparable to a nanoindenter setup. Conclusions Due to the flexibility of the method, it offers straightforward testing of cantilevers manufactured at arbitrary position on bulk samples with fully reversed plastic deformation. Specific microstructural features, e.g., selected orientations, grain boundaries, phase boundaries etc., can therefore be easily targeted.

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