Multi-isotope imaging mass spectrometry (MIMS) allows high-resolution quantitative imaging of protein and nucleic acid synthesis at the level of a single cell using stable isotope labels. We employed MIMS to determine the compartmental localization of selenoproteins tagged with stable isotope selenium compounds in human aortic endothelial cells (HAEC), and to compare the efficiency of labeling (to determine the ideal selenium source) from these compounds: [82Se]-selenite, [77Se]-seleno-methionine, and [76Se]-methyl-selenocysteine. We found that all three selenium isotopes appear to be localized in the nucleus as well as in the cytoplasm. For MIMS detection, we compared freeze-drying to thin layer versus thin sectioning for sample preparation. MIMS provides a unique and novel way to dissect selenoprotein synthesis in cells.