Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts

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

“…One significant limiting factor for whole-cell cryo-ET workflows has been obtaining sufficient numbers of collectable targets per grid. Recently, a number of groups have developed protocols for micropatterning grids for cryo-EM, with one advantage being improved data collection efficiency 16 , 17 , 18 . Here a protocol is presented for using a commercially available micropatterning system to micropattern TEM grids for cryo-ET studies of primary Drosophila neurons and cultured human cell lines (uninfected or RSV-infected).…”

“…Even on patterned grids, cells are often observed to be positioned in the corners of grid squares partially on both the patterned carbon foil and grid bar. Recently, micropatterning was used to intentionally position part of the cell over the grid bar 18 . This could be considered for experiments where it is not critical to have the entire cell periphery on the foil.…”

“…A single seeding step at an optimized cell density, as described in the protocol for Drosophila neurons, is a viable option for most cell types. However, it is also possible to seed cells onto the substrate at a lower initial concentration and add more cells in a monitored fashion as described here and in other literature 18 . This sequential seeding can provide more consistent results in some cases.…”

“…Micropatterned grids can be used to help position cells to establish a consistent cell density across the grid and to position regions of interest in areas suitable for tilt-series collection 16 , 18 . The placement and positioning of cells can be used as fiducial markers for correlation in cryo-CLEM experiments, reducing the need for fragile finder-grids and fluorescent fiducial markers.…”

“…ECM proteins can then be added to the patterns for the appropriate cell culture. This method has been used by several groups for cryo-ET studies of retinal pigment epithelial-1 (RPE1), Madin-Darby canine kidney-II (MDCKII), human foreskin fibroblast (HFF), and endothelial cell lines 16 , 17 , 18 . This micropatterning system is compatible with multiple anti-fouling layer substrates as well as either a liquid or gel photocatalyst reagent.…”