Thickness- and quality-controlled fabrication of fluorescence-targeted frozen-hydrated lamellae

The composition, conformation, and function of most macromolecular complexes depend on their cellular context and must be studied inside cells. Few cells are sufficiently thin to permit direct imaging with cryoelectron microscopy (cryo-EM). Focused ion beam milling enables cryo-EM to visualize macromolecules in cells at high resolution by generating thin sections of frozen-hydrated cells. We show how thin cellular sections can be prepared in a controlled fashion using an integrated light microscope coincident with electron and ion beams. The procedure provides live feedback on the thickness and uniformity of the prepared lamella, reducing complexity and increasing the success rate. Combined with its ability for fluorescence-based targeting, our procedure paves the way toward an automated workflow that allows for control over lamella quality, thickness, and target inclusion, facilitating the routine fabrication of frozen-hydrated cellular sections.