In atom probe tomography (APT), a technique that has been used to determine 3D maps of ion compositions of metals and semiconductors at sub-nanometer resolutions, controlled emissions of ions can be induced from needle-shaped specimens in the vicinity of a strong electric field. Detection of these ions in the plane of a position sensitive detector provides two-dimensional compositional information while the sequence of ion arrival at the detector provides information in the third dimension. Here we explore the use of APT technology for imaging biological specimens. We demonstrate that it is possible to obtain 3D spatial distributions of cellular ions and metabolites from unstained, freeze-dried mammalian cells. Multiple peaks were reliably obtained in the mass spectrum from tips with diameters of similar to 50 nm and heights of similar to 200 nm, with mass-to-charge ratios (m/z) ranging from 1 to 80. Peaks at m/z 12, 23, 28 and 39, corresponding to carbon, sodium, carbonyl and potassium ions respectively, showed distinct patterns of spatial distribution within the cell. Our studies establish that APT could become a powerful tool for mapping the sub-cellular distribution of atomic species, such as labeled metabolites, at 3D spatial resolutions as high as similar to 1 nm. Published by Elsevier Inc.