Correlative SBF-SEM Imaging of Cell-Cell Contacts

Prior to SBF-SEM, the block was sputter-coated with 5–10 nm platinum using a Q150R S sputter coater (Quorum Technologies). SBF-SEM data was collected using a 3View2XP (Gatan Inc.) attached to a Sigma VP SEM (Zeiss). The microscope was operated at 1.8kV with 30-μm aperture, using Focal Charge Compensation mode (Deerinck et al., 2018 (link)). Inverted backscattered electron images were acquired every 50 nm, at a resolution of 8.44 nm/pixel. Acquired images were imported into Fiji (Schindelin et al., 2012 (link)) and aligned using the Register Virtual Stack Slices (Arganda-Carreras et al., 2006 ) and in TrakEM2 using manual landmarks (Cardona et al., 2012 (link)). 63 × Z stack was matched to the aligned SBF-SEM data using BigWarp (Bogovic et al., 2016 ) based on nuclei positions and overlaid to the EM data after applying thin-plate spline transformation. Cells in the region of expanded cell-cell contacts were selected and segmented using the TrakEM2. Cell-cell contacts were segmented if the plasma membranes of two neighboring cells were closely apposed with spacing less than or equal to 25 nm based on the length of homo dimer of PECAM extracellular domains (Jiang et al., 2016 (link)). 3D reconstructions were made in the 3dmod program.