Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Nature Methods volume 17, pages 50–54 (2020)Cite this article

Subjects

Abstract

Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Micropatterning of cryo-EM grids refines preparation for cryo-FIB lamella micromachining from adherent mammalian cells.
Fig. 2: Cryo-EM grid micropatterning tailored for controlling cellular morphology and cytoskeletal architecture.

Similar content being viewed by others

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. The raw data are available from the corresponding author upon reasonable request.

References

  1. Kühlbrandt, W. The resolution revolution. Science 343, 1443–1444 (2014).

    Article  Google Scholar 

  2. Pfeffer, S. & Mahamid, J. Unravelling molecular complexity in structural cell biology. Curr. Opin. Struct. Biol. 52, 111–118 (2018).

    Article  CAS  Google Scholar 

  3. Beck, M. & Baumeister, W. Cryo-electron tomography: can it reveal the molecular sociology of cells in atomic detail. Trends Cell Biol. 26, 825–837 (2016).

    Article  Google Scholar 

  4. Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972 (2016).

    Article  CAS  Google Scholar 

  5. Schaffer, M. et al. Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. J. Struct. Biol. 197, 73–82 (2017).

    Article  CAS  Google Scholar 

  6. Rigort, A. et al. Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc. Natl Acad. Sci. USA 109, 4449–4454 (2012).

    Article  CAS  Google Scholar 

  7. Wang, K., Strunk, K., Zhao, G., Gray, J. L. & Zhang, P. 3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography. J. Struct. Biol. 180, 318–326 (2012).

    Article  CAS  Google Scholar 

  8. Hariri, H. et al. Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis. J. Cell Biol. 218, 1319–1334 (2019).

    Article  CAS  Google Scholar 

  9. Ader, N. R. et al. Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis. eLife 8, e40712 (2019).

    Article  Google Scholar 

  10. Khanna, K. et al. The molecular architecture of engulfment during Bacillus subtilis sporulation. eLife 8, e45257 (2019).

    Article  Google Scholar 

  11. Weiss, G. L., Kieninger, A. K., Maldener, I., Forchhammer, K. & Pilhofer, M. Structure and function of a bacterial gap junction analog. Cell 178, 374 e315–384.e15 (2019).

    Article  Google Scholar 

  12. Jasnin, M., Ecke, M., Baumeister, W. & Gerisch, G. Actin organization in cells responding to a perforated surface, revealed by live imaging and cryo-electron tomography. Structure 24, 1031–1043 (2016).

    Article  CAS  Google Scholar 

  13. Asano, S., Engel, B. D. & Baumeister, W. In situ cryo-electron tomography: a post-reductionist approach to structural biology. J. Mol. Biol. 428, 332–343 (2016).

    Article  CAS  Google Scholar 

  14. Förster, F. & Hegerl, R. Structure determination in situ by averaging of tomograms. Methods Cell Biol. 79, 741–767 (2007).

    Article  Google Scholar 

  15. Guo, Q. et al. In situ structure of neuronal C9orf72 poly-GA aggregates reveals proteasome recruitment. Cell 172, 696–705 (2018).

    Article  CAS  Google Scholar 

  16. Bäuerlein, F. J. B. et al. In situ architecture and cellular interactions of PolyQ inclusions. Cell 171, 179–187 (2017).

    Article  Google Scholar 

  17. Patla, I. et al. Dissecting the molecular architecture of integrin adhesion sites by cryo-electron tomography. Nat. Cell Biol. 12, 909–915 (2010).

    Article  CAS  Google Scholar 

  18. Marston, D. J. et al. High Rac1 activity is functionally translated into cytosolic structures with unique nanoscale cytoskeletal architecture. Proc. Natl Acad. Sci. USA 116, 1267–1272 (2019).

    Article  CAS  Google Scholar 

  19. Siegmund, S. E. et al. Three-dimensional analysis of mitochondrial crista ultrastructure in a patient with leigh syndrome by in situ cryoelectron tomography. iScience 6, 83–91 (2018).

    Article  CAS  Google Scholar 

  20. Sorrentino, S., Studt, J. D., Horev, M. B., Medalia, O. & Sapra, K. T. Toward correlating structure and mechanics of platelets. Cell Adh. Migr. 10, 568–575 (2016).

    Article  CAS  Google Scholar 

  21. Tamir, A. et al. The macromolecular architecture of platelet-derived microparticles. J. Struct. Biol. 193, 181–187 (2016).

    Article  CAS  Google Scholar 

  22. Hampton, C. M. et al. Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat. Protoc. 12, 150–167 (2017).

    Article  CAS  Google Scholar 

  23. Mader, A., Elad, N. & Medalia, O. Methods in enzymology. Methods Enzymol. 483, 245–265 (2010).

    Article  CAS  Google Scholar 

  24. Maimon, T., Elad, N., Dahan, I. & Medalia, O. The human nuclear pore complex as revealed by cryo-electron tomography. Structure 20, 998–1006 (2012).

    Article  CAS  Google Scholar 

  25. Azioune, A., Carpi, N., Tseng, Q., Thery, M. & Piel, M. Methods in cell biology. Meth. Cell Biol. 97, 133–146 (2010).

    Article  CAS  Google Scholar 

  26. Thery, M. Micropatterning as a tool to decipher cell morphogenesis and functions. J. Cell Sci. 123, 4201–4213 (2010).

    Article  CAS  Google Scholar 

  27. Strale, P. O. et al. Multiprotein printing by light-Induced molecular adsorption. Adv. Mater. 28, 2024–2029 (2016).

    Article  CAS  Google Scholar 

  28. Booy, F. P. & Pawley, J. B. Cryo-crinkling: what happens to carbon films on copper grids at low temperature. Ultramicroscopy 48, 273–280 (1993).

    Article  CAS  Google Scholar 

  29. Thery, M., Jimenez-Dalmaroni, A., Racine, V., Bornens, M. & Julicher, F. Experimental and theoretical study of mitotic spindle orientation. Nature 447, 493–496 (2007).

    Article  CAS  Google Scholar 

  30. Thery, M. et al. The extracellular matrix guides the orientation of the cell division axis. Nat. Cell Biol. 7, 947–953 (2005).

    Article  CAS  Google Scholar 

  31. Arnold, J. et al. Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy. Biophys. J. 110, 860–869 (2016).

    Article  CAS  Google Scholar 

  32. Engel, L. et al. Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies. J. Micromech. Microeng. 29, 115018 (2019).

    Article  Google Scholar 

  33. Thery, M. et al. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc. Natl Acad. Sci. USA 103, 19771–19776 (2006).

    Article  CAS  Google Scholar 

  34. Senger, F. et al. Spatial integration of mechanical forces by α-actinin establishes actin network symmetry. J. Cell Sci. https://doi.org/10.1242/jcs.236604 (2019).

    Article  CAS  Google Scholar 

  35. Jasnin, M. & Crevenna, A. H. Quantitative analysis of filament branch orientation in listeria actin comet tails. Biophys. J. 110, 817–826 (2016).

    Article  CAS  Google Scholar 

  36. Toro-Nahuelpan, M. et al. Prot. Exch. https://doi.org/10.21203/rs.2.12377/v1 (2019).

  37. Vignaud, T. et al. Reprogramming cell shape with laser nano-patterning. J. Cell Sci. 125, 2134–2140 (2012).

    Article  CAS  Google Scholar 

  38. Politi, A. Z. et al. Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging. Nat. Protoc. 13, 1445–1464 (2018).

    Article  CAS  Google Scholar 

  39. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  40. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  Google Scholar 

  41. Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to the Advanced Light Microscopy Facility and the cryo-Electron Microscopy Platform at the European Molecular Biology Laboratory (EMBL), especially to S. Terjung, A. Halavatyi, S. Schnorrenberg, W. Hagen and F. Weis, and to G. Celetti for the kind gift of purified GFP protein. The Alvéole Lab company members M. Akyuz, G. Peyret, L. Siquier and L. Bonnemay are acknowledged for advice and helpful discussions. J.M. is grateful to the EMBL for funding. M.T.-N. was supported by a fellowship from the EMBL Interdisciplinary (EI3POD) program under Marie Skłodowska-Curie Actions COFUND (no. 664726). This project received funding from the European Research Council: ERC 3DCellPhase- (grant no. 760067) to J.M., ERC ICEBERG (grant no. 771599) to M.T. and ERC AAA (grant no. 741773) to L.B.

Author information

Authors and Affiliations

  1. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Mauricio Toro-Nahuelpan, Ievgeniia Zagoriy & Julia Mahamid

  2. CytomorphoLab, Interdisciplinary Research Institute of Grenoble, Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble-Alpes/CEA/CNRS/INRA, Grenoble, France

    Fabrice Senger, Laurent Blanchoin & Manuel Théry

  3. CytomorphoLab, Hôpital Saint Louis, Institut Universitaire d’Hématologie, Université Paris Diderot, Paris, France

    Laurent Blanchoin & Manuel Théry

Authors
  1. Mauricio Toro-Nahuelpan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ievgeniia Zagoriy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fabrice Senger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laurent Blanchoin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Manuel Théry
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julia Mahamid
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.T.-N., L.B., M.T. and J.M. conceived the study. M.T.-N., M.T. and J.M. designed the experiments. M.T.-N., I.Z., F.S. and J.M. performed experiments. M.T.-N. and J.M. made the figures and wrote the manuscript, with contributions from all authors. M.T. and J.M. supervised the work.

Corresponding author

Correspondence to Julia Mahamid.

Ethics declarations

Competing interests

Authors are listed as inventors on a patent application relating to this report.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Micropatterning of electron microscopy grids for positioning of adherent cells at high spatial accuracy.

(a) Grid passivation with anti-fouling agent PLL-g-PEG generates an organized repulsive PEG brush at the surface. (b) UV laser application using a 355 nm pulsing laser scanned through the region of interest causes ablation of the passivation layer. (b’) UV laser application using a digital-micromirror device (DMD) with a Primo module and the photo-initiator (PLPP) to locally oxidize the passivation layer. (c) Spatially constrained ablation of the PLL-g-PEG passivation layer. (d) Grid functionalization with extracellular matrix (ECM)-related proteins. (e) Cell seeding at the functionalized micropatterned areas.

Supplementary Figure 2 Photo-micropatterning of EM grids using a UV-355 nm pulsing laser.

(a-b) Reflection and transmission light microscopy of micropatterns generated by a 355 nm wavelength pulsing laser scanned on the grid film to generate a 30 µm diameter disk-shaped area (gold-mesh grid, SiO2 film R1/4). The micropatterned area, imaged in the confocal microscope immediately post-laser application, can be identified by the impression left as a result of laser pulses on the SiO2 film. (c-d) Cryo-FIB/SEM imaging of a 40 µm disk-shaped micropatterned grid post-vitrification displaying the engraving made by the laser (gold-mesh grid, SiO2 film R1/4). (e) Gold-mesh holey (R1/4) SiO2 film grid micropatterned 4 x 4 grid squares (30 µm disk-shape. Patterned area delineated by yellow dashed line) around the grid center (cyan circle), treated with fibronectin and seeded with HeLa cells. Cells are constrained to the patterned area. (f) Light microscopy imaging of a grid 2 h after seeding (upper-panel), displaying single cells within the micropatterned circular region. Cell division (24 h post-seeding, lower-panel) is restricted to the micropatterned area. (g) FIB shallow angle view of a cell grown on a disk-shaped pattern under cryogenic conditions. Yellow rectangles indicate the pattern for milling to produce a thin lamella through the cell center. (h) FIB view of the cell after milling to generate a ~200 nm thin lamella. (i) SEM top view, and zoom-in (j), of the lamella from (h).

Supplementary Figure 3 Photo-micropatterning of various EM grids and film materials.

All displayed grids were micropatterned using a one-step passivation (unless stated otherwise) and the Primo™ module with a digital-micromirror device and the photo-initiator PLPP. (a-c) Gold-mesh grid with holey (R1/4) SiO2 film grid: (a) micropatterned 6 x 6 grid squares (H-shape of 30 µm size) around the grid center (cyan circle). The grid was incubated with GFP protein to validate micropatterning. Grid bars are also patterned (3 µm lines). Inset: cross-bow pattern generated following two-step passivation coated with fibrinogen-Alexa546 to validate micropatterning; (b-c) Grid with micropatterned (b) 8 x 7 (H-shape of 30 µm size) or (c) 6 x 6 grid squares (20 µm diameter disks) around the grid center, treated with fibronectin, seeded with HeLa cells and imaged 2 h post-seeding. (d) Gold-mesh coated with a holey (R2/1) amorphous carbon and an additional layer (2 nm thick) of continuous carbon. An area of 8 x 8 grid squares was micropatterned (20 µm diameter disks), and treated with GFP protein. (e) Titanium-mesh with a holey (R1.2/20) SiO2 film grid micropatterned with various shapes (35 µm size crosses, crossbows, and disks), coated with fibronectin, vitrified and imaged by low voltage (2 kV) cryo-SEM. (f-k) Gold-mesh with holey (R2/2) gold film. An area of 8 x 8 grid squares was micropatterned (20 µm diameter disks) and either (f) treated with GFP protein, or (g) treated with fibronectin, seeded with HeLa cells and imaged at 2 h post-seeding. (h) SEM image under cryogenic conditions of the same grid from (g). HeLa cells were allowed to adhere for five additional hours post-transfer to a cell-free dish (see methods) followed by plunge-freezing. (i) Cryo-SEM micrograph of the area framed in (h). (j) FIB shallow angle view under cryogenic conditions of the cells indicated in (i), grown on a disk-shaped pattern. (k) Cryo-SEM top view of the cell after milling to generate a thin lamella.

Supplementary Figure 4 Correlation of grid maps from fluorescence light microscopy and transmission electron microscopy on grids with complex micropatterns.

(a) On-grid live-cell imaging (confocal microscopy) to generate a grid map of RPE1 LifeAct-GFP culture (4h post-seeding) on gold-mesh (SiO2 film R1/4) grid with 8 x 7 patterned grid squares. Micrograph is a maximum intensity projection of a z-stack. (b) Fluorescence microscopy map from (a) overlaid with the pattern design. The micrograph is contrast adjusted for better visualization. (c) Cryo-TEM map of the same grid (vitrified ~1h after live-cell imaging), and overlaid with the fluorescence microscopy map. Cells are dark in the TEM map and light in the light microscopy map. Cyan circle: grid center. Micrographs are related to Fig. 2b and Supplementary Fig. 5.

Supplementary Figure 5 Cryo-ET of actin networks at the thin peripheries of RPE1 cells grown on complex micropatterns.

(a) Cryo-TEM map of gold-mesh, R1/4 SiO2 film grid with 8 x 7 patterned grid squares. The micrograph is rotated 135° counterclockwise relative to Fig. 2b and Supplementary Fig. 4. Framed cells are enlarged in (b), (e), (i), and (l). (b) Cryo-TEM micrograph of the grid square indicated in (a) grown on a cross-shaped pattern (same cell as in Fig. 2c). Yellow lines indicate the expected positioning of peripheral stress fibers as indicated from live cell imaging of the LifeAct fluorescence (Fig. 2a, top left). (c) Cryo-TEM micrograph of the framed area in (b), displaying a stress fiber targeted for tomography. (d) Tomographic slice (6.8 nm thickness) through the 3D volume of the specified area in (c), showing the parallel organization of actin filaments into a stress fiber. (e) Cryo-TEM micrograph of a different RPE1 cell grown on a cross-shaped pattern from the grid shown in (a). (f) Cryo-TEM micrograph of the framed area in (e), displaying a peripheral cell protrusion with intricate actin architecture. (g-h) Tomographic slices at two different z-positions (6.8 nm thickness) through the 3D volume of the specified area in (f), showing the organization of actin filaments into multiple inter-related bundles. (i) Cryo-TEM micrograph of an RPE1 cell grown on an oval-shaped pattern from the grid shown in (a). (j) Cryo-TEM micrograph of the framed area in (i), displaying the targeted area. (k) Tomographic slice (6.8 nm thickness) of the periphery of the cell depicting actin filaments bundling. (l) Cryo-TEM micrograph of RPE1 cell from the grid displayed in (a), spreading on an oval-shaped pattern. (m) Cryo-TEM micrograph of the framed area in (l), displaying the targeted area represented in Fig. 2f (frame).

Supplementary Figure 6 Actin network architecture in RPE1 cells grown on crossbow pattern.

(a) On-grid live-cell Airyscan confocal slice at the basal region of an RPE1 (LifeAct-GFP) cell (shown in Fig. 2a, top right), grown on a crossbow fibronectin-coated micropattern, and overlaid with the pattern design (yellow). (b) Schematic representation of the actin network organization throughout the whole cell correlates with physical cues provided by cell-shape restriction to the micropattern shape (in agreement with1): (i) an extended actin meshwork at the cellular periphery, corresponding to the edge of the arc, forms a lamellipodium in adhesive regions of the pattern. This network extends towards the inner part of the cell with (ii) perpendicular fibers and parallel arcs of filament bundles. (iii) Stress fibers connect regions of the cell positioned on non-adhesive passivated areas of the support. (c-e) Correlation of the actin network organization to cryo-ET data acquired on a cryo-FIB generated wedge from Fig. 2h. (c) SEM top view image of an RPE1 cell grown on a crossbow (yellow) fibronectin-coated micropattern. A wedge was produced to allow access to the inner basal part of the cell. Dashed blue line corresponds to the edge of the wedge generated and represented in (d). (d) After milling, a thin wedge at the basal cell membrane was generated by ablating the top of the cell. (e) Representative RPE1 cell from (a) grown on a crossbow micropattern displaying different architectures of the actin cytoskeleton (LifeAct-GFP) overlapped with the location of the wedge edge and tomograms positioning. Note, this is not a direct correlation, but only meant for illustration and localization purposes. (f) Schematic representation of the actin architecture as well as expected organelles positioning of RPE1 cells spread on a crossbow-shaped micropattern in accordance with1. Models are not to scale.

Supplementary Figure 7 Cryo-FIB milling followed by cryo-ET of a cell grown on complex micropattern.

(a) SEM imaging under cryogenic conditions of an RPE1 cell grown on a cross-shaped pattern (yellow; titanium-mesh grid with SiO2 film R1.2/20), and overlaid with the SEM micrograph of a wedge produced by cryo-FIB milling. The yellow square indicates the position of the tomographic slice in (d). (b) FIB shallow angle view of the cell shown in (a). Yellow rectangle indicates the pattern for milling aimed at ablating the top of the cell. (c) As a result of the cryo-FIB milling step, a thin wedge at the basal cell membrane is produced. (d) Tomographic slice of the position indicated by the yellow square in (a). Actin bundles equivalent to stress fibers (see Fig. 2a) are found in locations expected according to the actin map of a cross-shaped RPE1 cell.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and discussion.

Reporting Summary

Supplementary Video 1

Live-cell imaging of HeLa cells on micropatterned grids. Time-lapse, live-cell imaging of HeLa cells expressing GFP-tagged β-tubulin (cyan) and mCherry-tagged histone (H2B-mCherry: magenta) 8 h post-release from an S-phase cell-cycle block. Fields of view of a single grid square of a non-patterned (left) and micropatterned grid (right) are shown. Related to Fig. 1c,d. Gold grids with a SiO2 (R1.2/20) holey film. Scale, 20 µm.

Supplementary Video 2

Cryo-ET of a cell grown on a cross-shape micropattern. Tomographic volume of the thin periphery of an RPE1 cell grown on a cross-shape pattern. Conventional defocus tomogram denoised with an anisotropic nonlinear diffusion algorithm. Video related to Fig. 2e. Each slice is 6.8-nm thick. Scale, 100 nm.

Supplementary Video 3

Cryo-ET of a cell grown on an oval-shape micropattern. Tomographic volume of the thin periphery of an RPE1 cell grown on an oval-shape pattern. Conventional defocus tomogram. Video related to Fig. 2f. Each slice is 6.8-nm thick. Scale, 100 nm.

Supplementary Video 4

Cryo-ET of a cryo-FIB thinned cell grown on a crossbow-shape micropattern. Tomographic volume of a cryo-FIB generated wedge of an RPE1 cell grown on a crossbow-shape pattern. VPP defocus tomogram. Video related to Fig. 2j, wedge position 1. Each slice is 6.8-nm thick. Scale, 100 nm.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Toro-Nahuelpan, M., Zagoriy, I., Senger, F. et al. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nat Methods 17, 50–54 (2020). https://doi.org/10.1038/s41592-019-0630-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-019-0630-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing