Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Abstract

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

Figure 1:. General workflow of micropatterning for cryo-EM.

The workflow can be roughly divided four parts: Grid preparation, micropatterning, ECM and cell seeding, and cryo-preparation and data collection. Major steps of each section are listed below the headings and the approximate time to complete each section is shown to the left.

Figure 2:. Screen shot of the software with pattern positioned on grid.

Area 1 contains the μm/pix ratio for pattern design. Area 2 is the ruler for measuring a grid. Area 3 is where to add or change patterns and ROIs. Area 4 contains all of the information for pattern positioning and dose. Area 5 contains options for patterns, including toggling overlays, copying or deleting patterns, and selecting patterns for micropatterning. Area 6 is where templates can be saved and loaded. Larger views of areas 4 and 5 are shown below for clarity.

Figure 3:. RSV-infected BEAS-2B cells on the patterned cryo-TEM grid.

(A) Fluorescent image of the patterned grid after addition of fluorescently labeled ECM. The input pattern is shown in the lower left corner. (B) Brightfield image of BEAS-2B cells grown on the grid in A. (C) Merge of the image in A (cyan) and B (grey) with fluorescent image of RSV-infected cells (red) immediately prior to plunge-freezing; infected cells express mKate-2. Scale bars are 500 μm. (D) Low-magnification cryo-TEM map of the grid in B after plunge-freezing. Fluorescent images are pseudocolored. Scale bars are 500 μm.

Figure 4:. Live/Dead staining of patterned and unpatterned cells.

(A) Fluorescent image of HeLa cells grown on a patterned grid and stained with calcein-AM (live cell stain, green) and ethidium homodimer-1 (dead cell stain, red). (B) HeLa cells grown on an unpatterned grid and stained as in A. (C) Projection of confocal z-stacks of a HeLa cell on a patterned Quantifoil R2/2 grid with 0.01 mg/mL collagen and fibrinogen 647 ECM (red). Cell was stained with calcein-AM (green) and Hoechst-33342 (blue). (D) HeLa cells on unpatterned grid incubated with 0.01 mg/mL collagen and fibrinogen 647 ECM, incubated and stained with calcein-AM and Hoecsht-33342. The fluorescent images were merged with transmitted light (grayscale). (E) X,Z projection of C. (F) X,Z projection of D. Images are pseudocolored. Scale bars in (A) and (B) are 500 μm; scale bars in (C) - (F) are 10 μm.

Figure 5:. Cryo-ET of RSV-infected BEAS-2B cell on the patterned cryo-TEM grid.

(A) Cryo-EM grid square map of RSV infected BEAS-2B cell. Approximate cell boundary is indicated by the dashed green line. (B) Higher resolution image of area boxed in red in (A). Approximate cell boundary is indicated by dashed green line. RSV virions can be seen near the cell periphery (white arrow and yellow box). (C) Single z-slice from tomogram collected in the area of the yellow box in (B). Red arrows point to RSV F fusion protein, blue arrows point to the ribonucleoprotein (RNP) complex. The scale bars in (A)-(C) are embedded in the image.

Figure 6:. Primary neurons derived from the brains of 3^rd instar Drosophila melanogaster larvae on the patterned cryo-TEM grid.

(A) Overlaid live-cell fluorescence microscopy grid montage of Drosophila neurons expressing membrane-targeted GFP on patterned grid squares with 0.5 mg/mL fluorescent concanavalin A. Green: Drosophila neurons. Blue: Photopattern. (B) Cryo-EM image montage of the grid in (A) after cryo-preservation. Yellow circle notes the same grid square as in (A). (C) Cryo-EM image montage of the square highlighted by the yellow circle in (A) and (B). (D) Higher magnification image of the area bounded by the red circle in (C), where a tilt series was collected on the cell’s neurites. E. 25 nm thick slice of a tomogram reconstructed from the tilt series that was acquired from the red circle in (C). Various organelles can be seen in this tomogram, such as the mitochondria (cyan), microtubules (purple), dense core vesicles (orange), light vesicles (green), the endoplasmic reticulum (yellow), and actin (blue). Macromolecules, such as ribosomes (red), can also be seen in the upper right corner. Fluorescent images are pseudocolored. The scale bars in (A)-(E) are embedded in the image.

Figure 7:. Primary neurons derived from the brains of 3^rd instar Drosophila melanogaster larvae on unpatterned grids.

(A) Live-cell fluorescence microscopy grid montage of Drosophila neurons expressing membrane-targeted GFP on grid squares with 0.5 mg/mL concanavalin A. Green: Drosophila neurons. (B) Cryo-EM grid montage of the same grid in (A) after plunge-freezing. Yellow circle shows the same grid square as in (A). Note the presence of cellular debris and media contamination, which made target identification difficult compared to patterned grids. (C) Cryo-EM image montage of the square highlighted by the yellow circles in the (A) and (B) maps. (D) Higher magnification image of the area bounded by the red circle in (C), where a tilt series was collected on the cell’s neurites. (E) 25 nm thick slice of the reconstructed tomogram from the tilt series from (C) and (D). A number of organelles are visible in this tomogram, such as microtubules (purple), actin (blue), the endoplasmic reticulum (yellow), and dense core vesicles (orange). Macromolecules, such as ribosomes (red), can also be seen. Fluorescent images are pseudocolored. The scale bars in (A)-(E) are embedded in the image.

Figure 8:. Examples of possible problems with patterning.

Fluorescent images of labeled ECM deposited on micropatterned grids. (A) Uneven patterning across the grid due to uneven distribution of PLPP gel. (B) ECM cannot adhere to areas covered by the PDMS stencil during patterning. (C) Saturated gradient pattern (right side) or inverted pattern (left) on a grid patterned with too high total dose. (D) ECM is adhering to areas on the grid bars as well as patterned area due to reflections of the UV laser during patterning. Images are pseudocolored; input pattern is shown in the lower left; scale bars are 100 μm.