Simultaneous quantification of intracellular pH and lactate at the single-cell level using a genetically encoded fluorescence lifetime biosensor

Metabolism plays an essential role in supporting physiological functions and maintaining a stable intracellular environment, including pH homeostasis. It is of biological significance to dynamically track metabolites and pH simultaneously in living cells. Genetically encoded fluorescent protein biosensors are extensively used to monitor metabolites; however, their fluorescence intensity—the commonly used readout—is often responsive to pH fluctuations, rendering analyte quantification challenging. In this study, we report a novel method that involves the steady-state fluorescence, time-resolved fluorescence and occupancy of the biosensor, called biosensor occupancy-enabled absolute quantification (BOEAQ) method, for simultaneous measurement of pH and analyte concentrations. We first demonstrate that FiLa (a recently reported lactate biosensor) can serve as a high performance fluorescence lifetime biosensor for lactate with a large intensity-weighted lifetime response (∼1.1 ns and ∼1.2 ns at 405 nm and 470 nm excitation, respectively). In a proof-of-principle, we simultaneously quantify pH and lactate concentration with our BOEAQ method both in vitro and in living cells at the single-cell resolution. In principle, the BOEAQ methodology holds promise for applications to other metabolite biosensors, thereby expanding the possibilities for multiplexed imaging.