SignificanceThe molecular mechanisms of biological macromolecules and their assemblies are often studied using purified material. However, the composition, conformation, and function of most macromolecules depend on their cellular context, which must be studied inside cells. Focused-ion-beam (FIB) milling enables cryogenic electron microscopy to visualize macromolecules in cells at near atomic resolution by generating thin sections of frozen cells. However, the extent of FIB milling damage to frozen cells is unknown. Here, we show that Ga+FIB milling introduces damage to a depth of ~60 nm from each lamella surface, leading to a loss of recoverable information of up to 20% in 100 nm samples. FIB milling with Ga+therefore presents both an opportunity and an obstacle for structural cell biology.