Fig. 6: In situ cellular tomography of shape-guided thinned region of micropatterned adhering PTK-1 cell–cell contacts.

a Top view of a cryo-FIB-milled lamella across a vitrified PTK-1 cell–cell contact site imaged using a cryo-transmission electron microscope. The milling patterns above and below the sample were adjusted to leave a central ~200 nm thin slice (lamella) supported by the surrounding unmilled, vitrified material. The cuts below and above the lamella are for releasing stress introduced by the milling. Cell–cell contact regions are identified visually using the shape of the two interacting cells in cryo-SEM images (Fig. S12a, b illustrate shape-guided targeting of a region of interest). b Higher magnification of targeted region marked by the red box in (a) representing the area of tomogram acquisition. c 2-nm thick virtual slice through a tomogram of the lamella region marked by the red box in b, showing a clearly discernible cell–cell contact (marked by red box, P: plasma adjacent plasma membranes) between two adjacent cells. Other cellular features visible include dense packing of macromolecular complexes in the cytoplasm; R: ribosomes; ER: endoplasmic reticulum; M: mitochondrion; V: vesicle; LD: lipid droplet; N: the nuclear envelope separating the nucleoplasm from the cytoplasm and C: Chromatin within the nucleus. d 3D surface representations of segmented features. Plasma membranes in white, nuclear envelope in yellow, other membrane structures in orange, ribosomes in blue and actin filament assemblies in pink. The cell–cell contact is marked by a red box. A slice of the tomogram is shown in the background for reference. e 3D surface representation of adherens junctional punctum within cell–cell contact. Membrane is in white, connecting rods are in red. The inset shows an enlarged view of the boxed area in c. Scale bars in a = 5 μm; b = 500 nm, c = 250 nm.