Excitation-Dependent pKa Extends the Sensing Range of Fluorescence Lifetime pH Sensors.

Biological activity is strongly dependent on pH, which fluctuates within a variety of neutral, alkaline, and acidic local environments. The heterogeneity of tissue and subcellular pH has driven the development of sensors with different pKa values, and a huge assortment of fluorescent sensors have been created to measure and visualize pH in living cells and tissues. In particular, sensors that report based on fluorescence lifetime are advantageous for quantitation. Here, we apply a theoretical framework to derive how the apparent pKa of lifetime-based pH sensors depends on fluorescence excitation wavelength. We demonstrate that theory predicts the behavior of two different fluorescent protein-based pH sensors in solution as proofs-of-concept. Furthermore, we show that this behavior has great practical value in living cells because it extends the sensing range of a single sensor by simply choosing appropriate detection parameters to match the physiological pH range of interest. More broadly, our results show that the versatility of a single lifetime-based sensor has been significantly underappreciated, and our approach provides a means to use a single sensor across a range of pH environments.