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.
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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.
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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.
Authors are listed as inventors on a patent application relating to this report.
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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.
(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).
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).
(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 Figs. 1–7 and discussion.
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.
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.
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.
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.
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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
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