The application of focused ion beam (FIB) milling to frozen hydrated cells has made it possible to visualize thick cells and tissues with cryogenic electron microscopy (cryo-EM). However, standard protocols for Gallium liquid metal ion source (LMIS) and plasma FIB-milling cause damage extending ∼30–60 nm from each lamella surface, limiting the usable lamella volume. Limiting FIB damage is essential to realize the goals of in situ structural biology including unambiguously identifying single molecules in cells and determining the 3D structure of proteins to near-atomic resolutions. We sought to identify conditions that minimize FIB-damage in biological lamellae by using two-dimensional template matching (2DTM) to measure single molecule damage profiles and directly compare different ions and accelerating voltages. In contrast to prior reports we demonstrate that Xenon plasma FIB-milled lamellae show significantly reduced lamella damage relative to lamellae prepared with other ions. We further identify conditions to improve particle detection and increase signal in resulting lamellae by polishing with lower acceleration voltages. We show that the reduction in damage unlocks the benefit of thinner lamella.
Hall et al. (Sun,) studied this question.