scholarly journals Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography

2015 ◽  
Vol 25 (1) ◽  
pp. 49 ◽  
Author(s):  
Terence Wagenknecht ◽  
Chyongere Hsieh ◽  
Michael Marko
Keyword(s):  
Skeletal Muscle ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Great Promise ◽  
Cryo Electron Tomography ◽  
A New Technique ◽  
Triad Junction ◽  
Focused Ion Beam Milling

Cryo-electron tomography (cryo-ET) has emerged as perhaps the only practical technique for revealing nanometer-level three-dimensional structural details of subcellular macromolecular complexes in their native context, inside the cell. As currently practiced, the specimen should be 0.1- 0.2 microns in thickness to achieve optimal resolution. Thus, application of cryo-ET to intact frozen (vitreous) tissues, such as skeletal muscle, requires that they be sectioned. Cryo-ultramicrotomy is notoriously difficult and artifact-prone when applied to frozen cells and tissue, but a new technique, focused ion beam milling (cryo-FIB), shows great promise for “thinning” frozen biological specimens. Here we describe our initial results in applying cryo-FIB and cryo-ET to triad junctions of skeletal muscle.

scholarly journals Author response: Fully automated, sequential focused ion beam milling for cryo-electron tomography

2020 ◽  
Author(s):  
Tobias Zachs ◽  
Andreas Schertel ◽  
João Medeiros ◽  
Gregor L Weiss ◽  
Jannik Hugener ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Author Response ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

scholarly journals Multi-Scale 3D Cryo-Correlative Microscopy for Vitrified Cells

2020 ◽  
Author(s):  
Gong-Her Wu ◽  
Patrick G. Mitchell ◽  
Jesus G. Galaz-Montoya ◽  
Corey W. Hecksel ◽  
Emily M. Sontag ◽  
...  
Keyword(s):  
Large Scale ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
3D Visualization ◽  
Ion Beam ◽  
Three Dimensional ◽  
Yeast Cells ◽  
Multi Scale ◽  
Fluorescence Confocal Microscopy ◽  
Cryo Electron Tomography

SUMMARYThree-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

scholarly journals Opening Windows into the Cell: Focused-Ion-Beam Milling for Cryo-Electron Tomography

2014 ◽  
Vol 106 (2) ◽  
pp. 600a ◽  
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Jürgen M. Plitzko ◽  
Wolfgang Baumeister
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

scholarly journals Structural Cell Biology: Preparing Specimens for Cryo-Electron Tomography Using Focused-Ion-Beam Milling

2014 ◽  
Vol 20 (S3) ◽  
pp. 1222-1223
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Ben Engel ◽  
Jürgen Plitzko ◽  
Wolfgang Baumeister
Keyword(s):  
Cell Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Structural Cell ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

scholarly journals Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography

2020 ◽  
Vol 15 (6) ◽  
pp. 2041-2070 ◽  
Author(s):  
Felix R. Wagner ◽  
Reika Watanabe ◽  
Ruud Schampers ◽  
Digvijay Singh ◽  
Hans Persoon ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Whole Cells ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

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 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.

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