Calcium plays an important role in the physiology of bacterial cells and as a free soluble form inside the cells, it is tightly regulated at a low concentration. However, much higher amounts of stored calcium exist within cells and can be remobilized at diverse occasions. As a consequence, there has been a variety of techniques developed to quantitatively map the different forms of Ca in cells. Here we show how scanning transmission x-ray microscopy (STXM), a synchrotron-based microscopy technique, conducted in the soft x-ray range at the Ca L2,3 edges (340–360 eV) offers an original and informative quantitative view of different Ca reservoirs in bacterial cells at a spatial resolution better than 100 nm. To illustrate this, we analyzed mutants of the cyanobacterium Synechococcus elongatus PCC 7942, overexpressing different versions of a gene called ccyA from two phylogenetically distant cyanobacteria: Gloeomargarita lithophora and Synechococcus sp. PCC 6312. This gene is diagnostic of the capability of some cyanobacteria to form intracellular amorphous calcium carbonate (iACC) but its function remains unknown. Here we show that the overexpression of the ccyA gene in the iACC non-forming cyanobacterium S. elongatus PCC 7942 results in an increased Ca content, especially in some Ca-rich cells. Moreover, we show that STXM can discriminate four different reservoirs of Ca in cyanobacteria and provides quantitative assessment of their relative importance.