Sodium ions (Na+) are key regulators of molecular events in many cellular processes, yet the dynamics of this ion remain poorly defined. Developing approaches to identify and characterise Na+ microenvironments will enable more detailed elucidation of the mechanisms of signal transduction. Here we report the application of Spectral Phasor analysis to the Na+ fluorophore, CoroNa Green, to identify and spatially map spectral emissions that report Na+ microenvironments. We use differentiating stem cells where Na+ fluxes were reported as an antecedent. Myoblast stem cells were induced to differentiate by serum starvation and then fixed at intervals between 0 and 40-minutes of differentiation prior to addition of CoroNa Green. The fluorescent intensity was insufficient to identify discrete Na+ microenvironments. However, using Spectral Phasor analysis we identified spectral shifts in CoroNa Green fluorescence which is related to the Na+ microenvironment. Further, spectral-heterogeneity appears to be contingent on the distance of Na+ from the nucleus in the early stages of differentiation. Spectral Phasor analysis of CoroNa Green in fixed stem cells demonstrates for the first time that CoroNa Green has unique spectral emissions depending on the nature of the Na+ environment in differentiating stem cells. Applying Spectral Phasor analysis to CoroNa Green in live stem cells is likely to further elucidate the role of Na+ microenvironments in the differentiation process.