A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue (original) (raw)

Data availability

The in situ subtomogram averages of C. elegans ribosomes (EMD-4755 to EMD-4758), as well as the tomograms shown in Fig. 3 and Supplementary Fig. 5 (EMD- 4869 and EMD-4870), have been deposited in the Electron Microscopy Data Bank. Raw images can be requested from the corresponding authors upon reasonable request.

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Acknowledgements

We are grateful to G. Pfeifer, I. Wolf, T. Matthes-Roesler and R. Gatz for technical support. We thank A. Zinke and A. Hyman (MPI-CBG) for providing the worm lines, A. Klingl and G. Wanner (LMU) for access to the high-pressure freezing instrument and J. Arnold for assistance with the cryo-confocal imaging and correlation. J.M. was supported by postdoctoral research fellowships from EMBO and HFSP, and by the Weizmann Institute Women in Science Program. We also acknowledge support from the Deutsche Forschungsgemeinschaft Excellence Clusters CIPSM and SFB 1035, the Max Planck Society and Thermo Fisher Scientific Company.

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Author notes

  1. These authors contributed equally: S. Pfeffer, J. Mahamid, S. Kleindiek.

Authors and Affiliations

  1. Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Miroslava Schaffer, Tim Laugks, Sahradha Albert, Benjamin D. Engel, Wolfgang Baumeister & Juergen M. Plitzko
  2. Zentrum für Molekulare Biologie, Heidelberg University, Heidelberg, Germany
    Stefan Pfeffer
  3. Structural and Computational Biology Unit, EMBL, Heidelberg, Germany
    Julia Mahamid
  4. Kleindiek Nanotechnik GmbH, Reutlingen, Germany
    Stephan Kleindiek, Andreas Rummel & Andrew J. Smith

Authors

  1. Miroslava Schaffer
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  2. Stefan Pfeffer
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  3. Julia Mahamid
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  4. Stephan Kleindiek
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  5. Tim Laugks
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  6. Sahradha Albert
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  7. Benjamin D. Engel
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  8. Andreas Rummel
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  9. Andrew J. Smith
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  10. Wolfgang Baumeister
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  11. Juergen M. Plitzko
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Contributions

M.S., W.B. and J.M.P. conceived and supervised the study. J.M.P. acquired funding and administered the project. M.S. and S.K. designed and developed the technique with support from T.L., A.R. and A.J.S. J.M. maintained the worm culture and performed HPF, cryo-ultramicrotomy and cryo-FLM. M.S. performed the cryo-FIB lift-out with technical support from S.K., T.L. and A.J.S. M.S. performed the cryo-FIB sample preparation with contribution from J.M. for the cryo-FLM correlation. M.S., J.M. and B.D.E. performed the cryo-microscopy work. S.P. developed the pipeline for the ribosome analysis. S.P. performed the data analysis and visualization with contributions from S.A. M.S., S.P., B.D.E. and J.M.P. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence toMiroslava Schaffer or Juergen M. Plitzko.

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Competing interests

S.K. is managing director of Kleindiek Nanotechnik GmbH. A.J.S. and A.R. are employees of Kleindiek Nanotechnik GmbH. All other authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Instrument geometry during the lift-out preparation.

Schematic representation of the sample stage with the transfer shuttle, the cryo-gripper micromanipulator, the ion beam (IB, red) and the electron beam (EB, blue) in the FIB chamber, shown in isometric view (top) and side view (bottom) during the main steps of the preparation. The transfer shuttle has a pre-tilt of 45° and holds both the thick HPF bulk specimen and the thin TEM half-grid. The cryo-gripper is mounted with z-movement parallel and xy-movement perpendicular to the ion beam direction (red line), independent from stage rotation and tilt. It is aligned to the EB/IB coincidence point of the FIB. When not in use, it can be fully retracted. There are three main positions: A, Trench milling and lamella extraction from bulk HPF sample at 7° stage tilt and 0° stage rotation. The ion beam and gripping-plane are perpendicular to the HPF specimen surface. B, Cutting the lamella from the bulk material (except connecting bridges) at 28° stage tilt (the angle may vary, depending on the size of the lamella and trenches) and 180° stage rotation. The ion beam can access the bottom of the HPF specimen cross-section (lamella surface). The gripper is retracted to the parking position. C, Lamella insertion into the half-grid and final lamella thinning at 7° stage tilt and 180° stage rotation. The ion beam and the gripping-plane are parallel to the TEM half-grid surface and prepared slots.

Supplementary Figure 2 Lamella pockets fabricated by the FIB on a standard TEM half-grid.

A, SEM image of the half-grid with two sample pillars that have been modified by using ion beam milling and localized ion beam-induced Pt deposition at room temperature (‘OmniProbe’ half grids, Oxford Instruments NanoAnalysis, High Wycombe, UK). To produce the lamella pockets, first, two rectangle trenches are milled with a high current ion beam. Next, the slots are made by Pt deposition using four simple rectangle patterns. Finally, the slots are cleaned with a 5 nA ion beam. B,C, Magnified side (B) and top (C) views corresponding to the box indicated in A, showing a sample pillar with two lamella slots. D, Schematic representation of the modified TEM half-grid. E, Enlarged drawing of a specimen support pillar, in front and top views, showing areas of Pt deposition and milling. F, 3D rendering of a finished lamella pocket with a lamella inserted in the left slot position.

Supplementary Figure 3 The effect of conductive Pt coating on SEM imaging.

A, Two trenches have been milled around the area of interest with the ion beam perpendicular to the HPF specimen surface, and the lamella has been cut partly free with the ion beam (U-shape). In this step, the insulating frozen-hydrated material has been exposed and produces strong charging effects. B, After a conductive Pt sputter coating has been applied, charging is greatly reduced and imaging quality is improved. The experiment was repeated twice to obtain two ‘extraction-ready’ lamellae.

Supplementary Figure 4 Schematics of typical trench milling patterns with the ion beam perpendicular to the sample surface in order to produce a 3 µm thick volume of interest.

A, In the first step, two regular cross-section patterns (in yellow) are simultaneously milled on both sides of the region of interest using 1 nA beam current. The width of the pattern (24 µm) depends on the intended lamella width and should include approximately 10 µm excess width to accommodate the supporting area needed in the TEM half-grid as well as material loss during cutting. The height of the top pattern (8 µm) is required to allow insertion of the gripper. The height of the bottom pattern (20 µm) depends on the intended lamella height and must be large enough to allow a clear view of the cross-section (lamella surface) with the ion beam when cutting the lamella away from the bulk material. B, Next, both sides of the area are sequentially milled with a 0.5 nA beam current using a rectangular pattern and a tilt of 1° towards the lamella surface (darker regions indicate areas that have been milled). C, In a final step, both sides are milled with a tilt of 0.5° towards the lamella surface and a 0.3 nA beam current.

Supplementary Figure 5 Cryo-ET on a second FIB lamella of C. elegans tissue.

A, Cryo-TEM overview of a 150 nm thick cryo-FIB lamella prepared by the lift-out technique (2 tomograms were obtained from the lamella shown). B, x,y slice from a tomographic volume acquired at the framed area in A. C, Annotated representation of the tomogram in B. Ribosomes representing the p1-pre (blue), p1-post (yellow) and p2 classes (gray) were mapped back into the tomogram.

Supplementary Figure 6 Ribosome localization via template matching.

A, 500 ribosome-containing subtomograms were manually selected from one tomogram and iteratively aligned against a sphere as a starting reference (left). The subtomogram average converged to an 80S ribosome (right) within 10 iterations and could be subsequently used to generate a purely data-driven template for correlation-based ribosome localization. B, For template matching, the subtomogram average from A was downsampled to match the tomogram pixel size with 2x binning, and the 40S subunit head was removed using an ellipsoid mask as indicated by the blue sphere in A. This allows for efficient separation of true and false positives using subtomogram classification (see Supplementary Fig. 7a). C, Example cross-correlation function (red) obtained from template matching with the template shown in B. Putative ribosome positions are indicated by peaks in the cross-correlation function (red spots). Membrane-enclosed cellular compartments (gray) were superposed. D, Distribution of cross-correlation coefficients (CCC) for the 5000 highest-scoring peaks, which were extracted from the cross-correlation volume depicted in C while imposing a minimal Euclidean distance of 18.45 nm (9 voxels) between peaks. True and false positive matches were mostly separated and allowed for initial particle selection using a CCC cutoff, as indicated.

Supplementary Figure 7 Schematic illustration of the subtomogram analysis pipeline.

A, 13,688 aligned subtomograms retrieved by template matching were sorted into 5 classes. This allowed for separation of ribosome-containing subtomograms from false positives that lack the 40S subunit head not included in the template structure (upper row). Simultaneously, subtomograms were sorted into two different populations that were subsequently refined separately to 11.8 Å and 15.6 Å resolution (lower row). These populations mostly differ in the length of the resolved ribosomal RNA expansion segments, most prominently ES7L (red circle, see Supplementary Fig. 8). B, Further classification separated the ribosomes from population 1 into two different subpopulations that were refined separately to 13.7 Å and 12.3 Å resolution (lower row) and represent different states in the translational elongation cycle. Further classification of the ribosomes in population 2 did not yield structurally distinct subpopulations. Variability in resolution may be due to different tomogram acquisition schemes (see methods and Table S1). Alternatively, ribosomes contributing to population 2 might be translationally less active in general, which would also explain the absence of major subpopulations representing distinct translational elongation cycle states. C, Gold standard Fourier shell correlation (FSC) curves for ribosome population 1 (black), the pre- (red) and post-translocational states (blue) included in ribosome population 1, as well as ribosome population 2 (green). Resolution was determined using the FSC = 0.143 resolution criterion.

Supplementary Figure 8 Ribosome populations 1 and 2 differ in the length of the resolved surface-exposed eukaryote-specific ribosomal RNA expansion segments.

Difference density (red) between the two ribosome populations superposed on the subtomogram average of population 2 (gray). In the close-up views (bottom row), density for ribosome population 1 is superposed as blue mesh. Docking the atomic model of a mammalian 80S ribosome as a rigid body (light blue, PDB: 3j3f) reveals that the difference density almost exclusively co-localizes with surface-exposed eukaryote-specific ribosomal RNA expansion segments, including two arms of ES7L.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Supplementary Table 1 and Supplementary Protocol

Reporting Summary

Supplementary Video 1

The cryo-FIB lift-out procedure: a small volume of interest is extracted from a high-pressure frozen sample and, using a micromanipulator, transferred onto a TEM half-grid for final cryo-FIB thinning. Correlative cryo-FLM is first used to target the area of interest in the otherwise topographically smooth bulk sample surface, followed by trench milling to obtain a ‘extraction-ready’ lamella, lift-out, positioning in a half-grid, Pt-GIS deposition and final thinning to electron transparency.

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Schaffer, M., Pfeffer, S., Mahamid, J. et al. A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.Nat Methods 16, 757–762 (2019). https://doi.org/10.1038/s41592-019-0497-5

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