HPF and cryo-lamella preparation of CDMs for cryo-ET. (A) Schematic workflow of on-grid CDM vitrification: (1) sandwich assembly of on-grid CDMs, live-stained for collagen; (2) vitrification via HPF; (3) specimen recovery of (4) vitrified CDM. (B) All specimens are screened for their quality and to define regions of interest (ROI, annotated with a purple rectangle) by cryofluorescence light microscopy. A magnified image of the ROI, showing collagen fibers is depicted below. The reflected light is used to define landmarks for correlation, such as the milling window on the FIBSEM Autogrid, indicated by white arrows. (C) Cryo-lift-out FIBSEM workflow. Trenches are milled to isolate the lift-out (1), which is attached to a micromanipulator needle by redeposition milling (blue patterns, 2) and prior to cutting it loose from the bulk sample (red pattern, 2). The lift-out is extracted from the bulk sample (3) and attached to a finger-like protrusion on a half-moon grid by redeposition milling (4–5, blue patterns). The needle is cleanly pulled off and its attachment site is removed from the lift-out by FIB milling (red pattern) (6). The lift-out can be milled into a thin lamella (7–9) compatible with cryo-ET. Arrowheads indicate milling direction. Scale bar dimensions are 10 µm unless annotated otherwise.