Biological activity is strongly dependent on pH, which may fluctuate 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 developed 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 determine how the apparent pKa of lifetime-base pH sensors depends on fluorescence excitation wavelength. We then demonstrate that theory accurate predicts the behavior of two different fluorescent protein-base pH sensors. Furthermore, we show that this behavior has great practical value in matching the appropriate detection parameters with the physiological pH range. 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.
