Plant manganese (Mn) hyperaccumulation provides unusual insight into homeostasis of this essential micronutrient, in particular its excessive storage in shoot tissues. The compartmentation of hyperaccumulated foliar Mn appears exceptional among metal hyperaccumulators, since it occurs via specific microdistribution patterns. Here, three associated Mn hyperaccumulators, Virotia neurophylla, Maytenus fournieri, and Garcinia amplexicaulis exhibiting distinctly different Mn detoxification strategies were examined. Non-invasive sample preparation in conjunction with cryo scanning electron microscopy (SEM) was used to obtain in vivo quantitative microprobe X-ray and anatomical data from fully hydrated cells. Highly vacuolated large palisade mesophyll cells in V. neurophylla leaves were found to contain around 650 mM Mn. The large non-photosynthetic hypodermal cells of M. fournieri leaves, also with high vacuolar content, and the main site for Mn disposal, had an estimated mean vacuolar Mn concentration of around 600 mM. Previous qualitative X-ray mapping had shown Mn to be almost evenly sequestered across the entire leaf cross section of G. amplexicaulis. However, quantitative data obtained here showed a marked variation in localised concentrations that ranged between ~15 and >800 mM. Notable among these were mean values of >600 mM in spongy mesophyll cells, and ~800 mM within cells of a narrow sub epidermal layer preceding the palisade mesophyll. This study demonstrated the extraordinary Mn carrying capacities of different types of leaf cell vacuoles.