scholarly journals Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae

2019 ◽  
Author(s):  
Georg Wolff ◽  
Ronald W. A. L. Limpens ◽  
Shawn Zheng ◽  
Eric J. Snijder ◽  
David A. Agard ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Ion Beam ◽  
Phase Plate ◽  
Expansion Joints ◽  
Cellular Environment ◽  
Focussed Ion Beam ◽  
Technical Issues ◽  
Thin Electron ◽  
Thin Lamella

AbstractCryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from these forces, we milled “micro-expansion joints” alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-lamella bending in any biological sample milled on EM grids.

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.

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.

In situ Structure of Viral RNA by Cryo Electron Tomography with Volta Phase Plate, Energy Filtering and Direct Electron Counting

2016 ◽  
Vol 22 (S3) ◽  
pp. 74-75
Author(s):  
Z. Hong Zhou ◽  
Wong H. Hui ◽  
Jiayan Zhang ◽  
Ivo Atanasov ◽  
Cristina C. Celma ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Viral Rna ◽  
Phase Plate ◽  
Energy Filtering ◽  
Electron Counting ◽  
Cryo Electron Tomography

In-situ electrical characterisation of a photodiode during nano-structuring with a focussed ion beam

2012 ◽  
Vol 110 (4) ◽  
pp. 935-941
Author(s):  
Jan Junis Rindermann ◽  
Mohammed Henini ◽  
Pavlos G. Lagoudakis
Keyword(s):  
Ion Beam ◽  
Focussed Ion Beam

scholarly journals An in-situ approach for preparing atom probe tomography specimens by xenon plasma-focussed ion beam

2019 ◽  
Vol 202 ◽  
pp. 121-127 ◽  
Author(s):  
J.E. Halpin ◽  
R.W.H. Webster ◽  
H. Gardner ◽  
M.P. Moody ◽  
P.A.J. Bagot ◽  
...  
Keyword(s):  
Atom Probe Tomography ◽  
Ion Beam ◽  
Atom Probe ◽  
Focussed Ion Beam

scholarly journals Proteasomes tether to two distinct sites at the nuclear pore complex

2017 ◽  
Vol 114 (52) ◽  
pp. 13726-13731 ◽  
Author(s):  
Sahradha Albert ◽  
Miroslava Schaffer ◽  
Florian Beck ◽  
Shyamal Mosalaganti ◽  
Shoh Asano ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Cellular Environment ◽  
Subtomogram Averaging ◽  
Substrate Processing ◽  
Cellular Components ◽  
Transport Of Macromolecules

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

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.

scholarly journals Abstract P-3: The Structure of Self-Assembled Surfactant Micellar Networks by in situ Cryo-Electron Tomography

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S12-S12
Author(s):  
Yury Chesnokov ◽  
Andrey Shibaev ◽  
Roman Kamyshinsky ◽  
Vyacheslav Kralin ◽  
Olga Philippova ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Ion Beam ◽  
Phase 1 ◽  
Persistence Length ◽  
Wormlike Micelles ◽  
Second Phase ◽  
Dense Network ◽  
Cryo Electron Tomography ◽  
Self Assembled

Background: Surfactant molecules can form various self-assembled structures in aqueous solutions, including spherical and cylindrical micelles, lamellae, vesicles, etc. Elongated cylindrical (wormlike) micelles can entangle and form a dense network. The study of the un-perturbed native structure of wormlike micelles in such networks presents a great challenge, since the micelles are formed due to weak non-covalent interactions and may easily break when external conditions are changed. In this work in situ cryo-electron tomography (cryo-ET) was applied to reveal the relaxed structure of such entangled systems. Methods: To prepare samples for the cryo-ET study 1 µl of the aqueous surfactant-containing solution was applied to the glow discharged grid, blotted with filter paper for 10 sec, drained for 60 sec to allow for the relaxation of the system and plunge-frozen with Vitrobot Mark IV. The vitrified sample was transferred to Versa 3D cryo-focused ion beam / scanning electron microscope (cryo-FIB/SEM) to prepare thin (100-150 nm) sections of the sample. Cryo-ET study was conducted using Titan Krios. IMOD and Avizo software packages were used for data processing. Results: In this work, wormlike micelles formed by a mixture of an anionic and a cationic surfactant were investigated at the excess of the anionic surfactant. Cryo-ET study of the obtained lamellae demonstrated the formation of two different phases, consisting of straight rods oriented along the grid substrate (phase 1) and isotropic network formed by wormlike micelles (phase 2) above it. The topology of the second phase corresponded to the branched saturated network or entangled network depending on cation/anion ratio of the sample. However, the analysis of the thin samples obtained without cryo-FIB demonstrated only the presence of the metastable phase (phase 1), which could lead to false conclusions regarding the morphology of the micelles. Conclusion: Here we discuss the influence of different sample preparation approaches on the sample structure and demonstrate that the native un-perturbed conformation of charged cylindrical surfactant micelles in the dense network is that of a slightly bent rod or a wormlike chain with high persistence length.

scholarly journals PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergey Gorelick ◽  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Travis K Johnson ◽  
Ava Handley ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Fluorescent Proteins ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Ion Beam ◽  
Correlative Microscopy ◽  
New Approach ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Ion microprobe analysis—a review of geological applications

1989 ◽  
Vol 53 (369) ◽  
pp. 3-24 ◽  
Author(s):  
S. J. B. Reed
Keyword(s):  
Microprobe Analysis ◽  
Ion Beam ◽  
Isotopic Analysis ◽  
Specimen Preparation ◽  
Ion Microprobe ◽  
Spatially Resolved ◽  
Secondary Ions ◽  
Focussed Ion Beam ◽  
Isotope Studies

AbstractIn ion microprobe analysis the specimen is bombarded with a focussed ion beam a few µm in diameter and the secondary ions produced are accelerated into the entrance slit of a mass spectrometer. An outline of the salient features of the instrument is given here, together with an account of the methods used for quantitative elemental and isotopic analysis.The major part of this paper consists of a comprehensive account of the geological applications of ion microprobe analysis. These include elemental analysis, especially for trace elements (down to sub-ppm levels in many cases) and light elements (H-F) which are beyond the scope of the electron microprobe. The other main area of geological interest is isotopic analysis, where the ion microprobe has the advantage over conventional mass spectrometry of being capable of in situ analysis of selected points on polished sections, obviating the need for laborious specimen preparation, and enabling spatially-resolved data to be obtained, with a resolution of a few µm. The ion microprobe has been especially successful in U-Pb zircon dating and the study of isotope anomalies in meteorites. Other significant applications include diffusion and stable isotope studies.

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