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We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission  in vitro . When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification  in vivo . Its spectral ratio response is H +  specific; neither Ca ++ , Mg ++ , Na + , nor K +  changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H +  ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH