Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

l-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics of l-Lactate, we develop a second-generation green fluorescent extracellular l-Lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular l-Lactate biosensor, designated R-iLACCO1. Compared to the first-generation eLACCO1.1 (Δ F / F = 1.5 in cultured neurons), eLACCO2.1 exhibits better membrane localization and fluorescence response (Δ F / F = 8.1 in cultured neurons) with faster response kinetics to extracellular l-Lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes in l-Lactate concentration in vitro (Δ F / F = 15 to 22) and in live mammalian cells (Δ F / F = 5.5 to 11). We demonstrate that these biosensors enable cellular-resolution imaging of extracellular and intracellular l-Lactate in cultured mammalian cells. ### Competing Interest Statement The authors have declared no competing interest.