A two-photon fluorescent probe records the intracellular pH through ‘OR’ logic operation via internal calibration

Mapping the intracellular location and concentration of hydronium ions (H₃O⁺) and their dynamics could be a useful diagnostic tool in modern clinical science. Current needs motivated us to develop a molecular pH probe 1, operating as a logic gate, and its analogue 2. The pyridyl moiety in 1 played a significant role in proton capture and release, in acidic to alkaline pH environments. In contrast, 2 failed to show a similar spectroscopic behavior. 1 shows emission maximum at 450 nm that is independent on the pH, with excitation at 353 nm or 410 nm in acid and alkaline pH, respectively. 1 was employed to provide input-dependent (excitation wavelength) fluorescence images in a cellular milieu to detect pH changes in cellular organelles such as lysosomes and mitochondria. Furthermore, 1 provided information on the variation of the pH in the presence of cellular ROS. 1 was also found to enable the real-time monitoring of cell acidification due to nutrient starvation, which is closely associated with mitochondrial malfunction, fusion and mitophagy processes. We envision that in due course 1 can open up new research avenues in the diagnostic sector for validating the pH in the cellular milieu.