The analysis of a cell with dynamic SIMS ion microscopy depends on the gradual erosion (sputtering) of the cell surface for obtaining spatially resolved chemical information in the X-, Y-, and Z-dimensions. This ideal feature of ion microscopy is rarely explored in probing microfeatures hidden beneath the cell surface. In this study, this capability is explored for the analysis of cells undergoing cell division. The mitotic cells required 3D SIMS imaging in order to study the chemical composition of specialized subcellular regions, like the mitotic spindle, hidden beneath the cell surface. Human glioblastoma T98G cells were grown on silicon chips and cryogenically prepared with a sandwich freeze-fracture method. The fractured freeze-dried cells were used for SIMS analysis with the microscope mode of the CAMECA IMS-3f, which is capable of producing 500 nm lateral image resolution. SIMS analysis of calcium in the spindle region of metaphase cells required sequential recording of as many as 10 images. The T98G human glioblastoma tumor cells revealed an unusual depletion/lack of calcium store in the metaphase spindle, which is in contrast to the accumulation of calcium stores generally observed in normal cells. This study shows the feasibility of the microscope mode imaging in resolving subcellular microfeatures in 3D and opens new avenues of research in spatially resolved chemical analysis of dividing cells. (C) 2004 Elsevier B.V. All rights reserved.