A quiet revolution is under way in technologies used for nanoscale cellular imaging. Focused ion beams, previously restricted to the materials sciences and semiconductor fields, are rapidly becoming powerful tools for ultrastructural imaging of biological samples. Cell and tissue architecture, as preserved in plastic-embedded resin or in plunge-frozen form, can be investigated in three dimensions by scanning electron microscopy imaging of freshly created surfaces that result from the progressive removal of material using a focused ion beam. The focused ion beam can also be used as a sculpting tool to create specific specimen shapes such as lamellae or needles that can be analyzed further by transmission electron microscopy or by methods that probe chemical composition. Here we provide an in-depth primer to the application of focused ion beams in biology, including a guide to the practical aspects of using the technology, as well as selected examples of its contribution to the generation of new insights into subcellular architecture and mechanisms underlying host-pathogen interactions.