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

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.

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

eLife ◽

10.7554/elife.04889 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 110

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.

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

10.1101/2021.12.31.474669 ◽

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

European Journal of Translational Myology ◽

10.4081/ejtm.2015.4823 ◽

2015 ◽

Vol 25 (1) ◽

pp. 49 ◽

Cited By ~ 15

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.

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

10.7554/elife.52286.sa2 ◽

2020 ◽

Cited By ~ 1

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

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The structure of the COPI coat determined within the cell

eLife ◽

10.7554/elife.32493 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 60

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.

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The molecular architecture of engulfment during Bacillus subtilis sporulation

eLife ◽

10.7554/elife.45257 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Bacillus Subtilis ◽

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

High Spatial Resolution ◽

Electron Tomography ◽

Ion Beam ◽

Molecular Architecture ◽

Native State ◽

Cryo Electron Tomography

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

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Decision letter: Fully automated, sequential focused ion beam milling for cryo-electron tomography

10.7554/elife.52286.sa1 ◽

2019 ◽

Keyword(s):

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Cryo Electron Tomography ◽

Focused Ion Beam Milling

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

eLife ◽

10.7554/elife.45919 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 20

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.

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Protocol for cryo-focused ion beam lift-out technique

10.21203/rs.2.10392/v1 ◽

2019 ◽

Cited By ~ 1

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.

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