Mitochondrial membrane potential, an indicator of mitochondrial function, is perturbed in many diseases, including cancer and neurodegenerative diseases, but difficult to measure noninvasively. Now, Elena A. Goun of the Swiss Federal Institute of Technology, Lausanne (EPFL) and coworkers have devised a bioluminescent probe for monitoring mitochondrial membrane potential (Nat. Chem. Biol. 2020, DOI: 10.1038/s41589-020-0602-1). The probe comprises two parts, a triphenylphosphine-caged luciferin and a separate azido-triphenylphosphine. Both components use their triphenylphosphonium groups to target mitochondria, where they react via Staudinger ligation, uncaging the luciferin in the process. The luciferin is acted on by the enzyme luciferase, which the researchers engineer into cells and mice. The enzymatic reaction releases light, the intensity of which is proportional to the concentration of the two parts of the probe. The rate enhancement of the reaction in the mitochondria relative to the rest of the cell enables measurement of the mitochondrial membrane potential. The researchers used the probe to measure age-related changes in mitochondrial membrane potential and showed that nicotinamide riboside could reverse the changes. They also used the probe to measure mitochondrial membrane potential in cancer in mice before and after the administration of nigericin, an antibiotic known to modulate mitochondrial membrane potential.