The reduction of FIB damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis

Focused ion beam (FIB) is widely used for thinning frozen cells to produce lamellae for cryo-electron microscopy imaging and for protein structures study in vivo. However, FIB damages the lamellae and a quantitative experimental analysis of the damage is lacking. We used a 30-keV gallium FIB to prepare lamellae of a highly concentrated icosahedral virus sample. The viruses were grouped according to their distance from the surface of lamellae and reconstructed. Damage to the approximately 20-nm-thick outermost lamella surface was similar to that from exposure to 16 e−/Å2 in a 300-kV cryo-electron microscope at high-resolution range. The damage was negligible at a depth beyond 50 nm, which was reduced to 30 nm if 8-keV Ga+ was used during polishing. We designed extra steps in the reconstruction refinement to maximize undamaged signals and increase the resolution. The results demonstrated that low-energy beam polishing was essential for high-quality thinner lamellae. [Display omitted] •A quantitative analysis method of cryo-FIB damage using virus lamellae is presented•Cryo-EM lamellae milled by 30-keV gallium FIB contained a 50-60-nm damage layer•Low energy milling reduced cryo-FIB damage and increased the quality of lamellae•The quality of reconstruction improved by a depth-dependent weighting function Yang et al. developed a quantitative analysis method of cryo-FIB damage using high-concentrated virus sample. The damage depth of 30-keV gallium FIB was estimated as 50 nm. Cryo-lamellae quality can benefit from low energy milling and the resolution can be increased by a depth-dependent weighting function.