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Intracellular potassium and calcium plays a crucial role in maintaining the membrane potential to the functioning cells, and the irregular changes of their concentrations can induce severe clinical problems (e.g., cardiac arrhythmias, nerve dysfunction, kidney failure). Therefore, simultaneous assessing the intra-cellular potassium and calcium concentrations is clinically desired. Such assessments would benefit from tools which can perform high resolution imaging measurements noninvasively in vivo. Here we developed a high resolution fluorescence imaging technique to simultaneously image intra-cellular potassium and calcium concentrations from tissue level down to cellular resolution, and more importantly, with time-lapse imaging technique, we can track the dynamic concentration changes in response to functional or pharmacological stimuli in vivo.