For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high-resolution imaging techniques. In the case of bone, focused ion beam-scanning electron microscopy (FIB-SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin-embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB-SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization.