scholarly journals Automated cryo-lamella preparation for high-throughput in-situ structural biology

2019 ◽  
Author(s):  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Cyntia Taveneau ◽  
Hari Venugopal ◽  
James C. Whisstock ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Automated Method ◽  
Cellular Environment ◽  
Transmission Electron

AbstractCryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Protocol for cryo-focused ion beam lift-out technique

2019 ◽  
Author(s):  
Miroslava Schaffer ◽  
Stefan Pfeffer ◽  
Julia Mahamid ◽  
Stephan Kleindiek ◽  
Tim Laugks ◽  
...  
Keyword(s):  
High Pressure ◽  
Structural Biology ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Electron Tomography ◽  
Ion Beam ◽  
Cell Interior ◽  
Cryo Electron Tomography ◽  
Multicellular Organisms

Abstract Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

scholarly journals In situ structural analysis of Golgi intracisternal protein arrays

2015 ◽  
Vol 112 (36) ◽  
pp. 11264-11269 ◽  
Author(s):  
Benjamin D. Engel ◽  
Miroslava Schaffer ◽  
Sahradha Albert ◽  
Shoh Asano ◽  
Jürgen M. Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Asymmetric Membrane ◽  
Protein Arrays ◽  
Cellular Environment ◽  
Filament Bundles ◽  
Subtomogram Averaging ◽  
And Function

We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function.

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.

Formation of Sub-100 NM GE Wires on Si by E-Beam Evaporation/Lithography

1995 ◽  
Vol 380 ◽  
Author(s):  
C. Deng ◽  
J. C. Wu ◽  
C. J. Barbero ◽  
T. W. Sigmon ◽  
M. N. Wybourne
Keyword(s):  
Cross Section ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Si Substrates ◽  
Novel Technique ◽  
Atomic Force ◽  
Transmission Electron ◽  
First Time ◽  
Scanning Electron

ABSTRACTA fabrication process for sub-100 nm Ge wires on Si substrates is reported for the first time. Wires with a cross section of 6 × 57 nm2 are demonstrated. The wire structures are analyzed by atomic force (AFM), scanning electron (SEM), and transmission electron microscopy (TEM). Sample preparation for TEM is performed using a novel technique using both pre and in situ deposition of multiple protection layers using a Focused Ion Beam (FIB) micromachining system.

scholarly journals Sample Preparation Methodologies for In Situ Liquid and Gaseous Cell Analytical Transmission Electron Microscopy of Electropolished Specimens

2016 ◽  
Vol 22 (6) ◽  
pp. 1350-1359 ◽  
Author(s):  
Xiang Li Zhong ◽  
Sibylle Schilling ◽  
Nestor J. Zaluzec ◽  
M. Grace Burke
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Focused Ion Beam ◽  
General Procedure ◽  
Ion Beam ◽  
Metals And Alloys ◽  
Stainless Steel Sample ◽  
Transmission Electron ◽  
Wide Range

AbstractIn recent years, an increasing number of studies utilizing in situ liquid and/or gaseous cell scanning/transmission electron microscopy (S/TEM) have been reported. Because of the difficulty in the preparation of suitable specimens, these environmental S/TEM studies have been generally limited to studies of nanoscale structured materials such as nanoparticles, nanowires, or sputtered thin films. In this paper, we present two methodologies which have been developed to facilitate the preparation of electron-transparent samples from conventional bulk metals and alloys for in situ liquid/gaseous cell S/TEM experiments. These methods take advantage of combining sequential electrochemical jet polishing followed by focused ion beam extraction techniques to create large electron-transparent areas for site-specific observation. As an example, we illustrate the application of this methodology for the preparation of in situ specimens from a cold-rolled Type 304 austenitic stainless steel sample, which was subsequently examined in both 1 atm of air as well as fully immersed in a H2O environment in the S/TEM followed by hyperspectral imaging. These preparation techniques can be successfully applied as a general procedure for a wide range of metals and alloys, and are suitable for a variety of in situ analytical S/TEM studies in both aqueous and gaseous environments.

The role of ion-beam cleaning in the growth of strained-layer epitaxial thin transition metal films

1987 ◽  
Vol 2 (4) ◽  
pp. 446-455 ◽  
Author(s):  
Sung I. Park ◽  
A. Marshall ◽  
R. H. Hammond ◽  
T. H. Geballe ◽  
J. Talvacchio
Keyword(s):  
Tunnel Junctions ◽  
Ion Beam ◽  
Surface Damage ◽  
Metal Films ◽  
Strained Layer ◽  
Transmission Electron ◽  
Lattice Matched

Low-energy ion-beam cleaning of the substrates prior to a deposition greatly enhances the quality of ultrathin (< 100 Å) refractory superconducting (Nb, V) films. Using this technique Nb films as thin as 7 Å have been grown, from which good tunnel junctions have been fabricated. Both the native films and the tunnel junctions are sturdy and can be thermally recycled without any degradation. In-situ surface study along with transmission electron microscopy (TEM) results suggest the removal of the carbon atoms from the surface of the substrate without an apparent surface damage as the causes of the improvement. The TEM results indicate that the Nb films grow perfectly lattice matched to the sapphire substrate when the substrate is ion-beam cleaned. This strained-layer epitaxy is observed up to 40 Å, the maximum thickness investigated through TEM.

scholarly journals Computer-Controlled Polishing System For Preparing Multiple Pre-FIB TEM Specimens

2007 ◽  
Vol 15 (6) ◽  
pp. 38-39
Author(s):  
D. J. MacMahon ◽  
E. Raz-Moyal
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Product Yield ◽  
Interface Layer ◽  
Ex Situ ◽  
Advantages And Disadvantages ◽  
Transmission Electron ◽  
Invaluable Tool ◽  
Electron Microscopes

Semiconductor manufacturers are increasingly turning to Transmission Electron Microscopes (TEMs) to monitor product yield and process control, analyze defects, and investigate interface layer morphology. To prepare TEM specimens, Focused Ion Beam (FIB) technology is an invaluable tool, yielding a standard milled TEM lamella approximately 15 μm wide, 5 μm deep and ~100 nm thick. Several techniques have been developed to extract these tiny objects from a large wafer and view it in the TEM. These techniques, including ex-situ lift-out, H-bar, and in-situ lift-out, have different advantages and disadvantages, but all require painstaking preparation of one specimen at a time.

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yury S Bykov ◽  
Miroslava Schaffer ◽  
Svetlana O Dodonova ◽  
Sahradha Albert ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Model ◽  
Electron Tomography ◽  
Ion Beam ◽  
Membrane Thickness ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

Growth of MBE-Codeposited IrSi3 on Si(111) and Si(100)

1994 ◽  
Vol 299 ◽  
Author(s):  
Gary A. Gibson ◽  
Davis A. Lange ◽  
Charles M. Falco
Keyword(s):  
Ion Beam ◽  
Silicide Phase ◽  
X Ray Diffraction ◽  
X Ray ◽  
Low Energy Electron Diffraction ◽  
Growth Modes ◽  
Transmission Electron ◽  
Low Energy Electron

AbstractWe have used Molecular Beam Epitaxy (MBE) to successfully grow films that are predominantly IrSi3 on both Si(111) and Si(100) substrates by codeposition of Si and Ir in a 3:1 ratio. Bragg-Brentano and Seemann-Bohlin x-ray diffraction reveal that polycrystalline IrSi3 films form as low as 450 °C. This is the lowest temperature yet reported for growth of this iridium silicide phase. These x-ray diffraction techniques, along with Transmission Electron Microscope (TEM) diffraction and in situ Low Energy Electron Diffraction (LEED), show that at higher deposition temperatures codeposition can form IrSi3 films on Si(111) that consist predominantly of a single epitaxial growth orientation. Ion beam channeling and x-ray rocking curves show that the epitaxial quality of IrSi3 films deposited on Si(111) is superior to that of IrSi3 films deposited on Si(100). We also present evidence for several new epitaxial IrSi3 growth modes on Si(111) and Si(100).

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