Adenosine 5'-triphosphate (ATP) is the major energy currency of all living organisms. Despite its important 4 functions, the spatiotemporal dynamics of ATP levels inside 3 living multicellular organisms is unclear. In this study, we 2 modified the genetically encoded Forster resonance energy transfer (FRET)-based ATP biosensor ATeam to optimize its affinity at low temperatures. This new biosensor, AT1.03NL, detected ATP changes inside Drosophila S2 cells more sensitively than the original biosensor did, at 25 degrees C. By expressing AT1.03NL in Drosophila melanogaster and Caenorhabditis elegans, we succeeded in imaging the in vivo ATP dynamics of these model animals at single-cell resolution.