This study investigates the metal content and compartmentalization changes in whole cells of diatom Coscinodiscus eccentricus exposed to metal overload, examining consequences to cellular toxicity, tolerance mechanisms, and metal fate in the environment. Cells exposed to Ni, Cu and Zn were analysed using nuclear microprobe techniques. Particle induced X-ray emission (PIXE), Rutherford backscattering spectrometry (RBS), and scanning transmission ion microscopy (STIM) were used simultaneously to obtain high-resolution imaging of morphological and quantitative elemental distribution data. Elemental partitioning within cell compartments, such as cell wall, cytoplasm and major organelles, was assessed. Diatoms clearly responded to excess metal levels, by changing cytoplasm morphology, concentrating added metals, and altering Fe transport mechanisms. Different metal accumulation patterns indicated high susceptibility to Cu, retained in the cytoplasm, and detoxification capability for Ni and Zn, mobilized to the vacuole. Iron and Zn were accumulated in the siliceous wall. Different metal distributions within the cell imply distinct environmental fates, Cu and Ni remain available with potential for biomagnification through the food web, whereas Fe and Zn are deposited at the bottom through frustule sedimentation.