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. A probe reports absolute sodium levels in acidic organelles with single-organelle resolution.