FIGURE 2:

Cryo-MATE workflow validation: the preparation modules are compatible with cell adhesion, growth, imaging, vitrification, and cryo-EM/ET. We used MCF-10A cells expressing a farnesylated form of EGFP for cell surface demarcation and tested two conditions to validate our workflow: a benchmark condition, A–F, with cells plated on the bare carbon film of the EM grid and a modified condition, G–L, with cells plated on a rBM-coated grid placed on top of a compliant (75-Pa) hydrogel and subjected to treatment with the myosin II inhibitor Blebbistatin. The blue squares in A and G indicate the cell chosen for the validation experiment (cells in Embra grid positions B12 and E2, respectively). CLEM images were collected and compared for each cell (B, H, fluorescence images; C, I, cryo-EM images; D, J, overlay of the fluorescence and cryoEM images to help delineate the cell area displaying the EGFP reporter; E, K, cryo-EM overviews of the cell area chosen for cryo-ET (white asterisks in E and K mark the position of the cell body); F, L, 2 nm–thick slice from a tomogram taken of the area of interest. As shown in the images, the grid and cells in the modified condition behaved similarly to those in the benchmark condition, with no loss in carbon-layer integrity, cell adhesion, or preservation of the ultrastructure. Scale bars, A, G, 40 µm; B, C, D, H, I, J, 10 µm; E, K, 1 µm; F, I, 200 nm.