Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+homeostasis is largely limited to Na+transport at the plasma membrane. Organelles may also contribute to Na+homeostasis; however, the direction of Na+flow across organelle membranes is unknown because organellar Na+cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+at single endosome resolution in mammalian cells and Caenorhabditiselegans, we discovered that lumenal Na+levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+levels in nematodes are modulated by the Na+/H+exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+will unveil mechanisms of Na+homeostasis at an increased level of cellular detail.