Real-Time Monitoring of Mitochondrial pH in HeLa Cells, Drosophila melanogaster, and Zebrafish Larvae Using BODIPY-Based Ratiometric Fluorescent Probes.

Three BODIPY-based fluorescent probes, AH⁺, BH⁺, and CH⁺, were synthesized for ratiometric pH sensing in living cells, fruit flies, and zebrafish larvae. These probes were designed to target mitochondrial environments by functionalizing the BODIPY core with various substituent groups that tune the pH sensitivity. The probes exhibited strong ratiometric fluorescence changes with pK_a values (AH⁺, 7.3; BH⁺, 7.5; CH⁺, 7.2) suitable for mitochondrial pH detection. Theoretical calculations supported these findings by establishing the geometries and electronic transitions and also resulted in the derivation of their pK_a values. Confocal imaging confirmed mitochondrial accumulation of these probes in HeLa cells, facilitating broad-range pH monitoring across a wide pH spectrum (3.5 to 9.1). These ratiometric pH sensors display good reversibility and response times under varying pH conditions. In application, the probe AH⁺ was employed to monitor pH fluctuations under conditions of oxidative stress and nutrient deprivation. Dual-channel cell imaging revealed a pH-dependent fluorescence shift with precise transitions, demonstrating the feasibility of real-time monitoring of the mitochondrial membrane potential in living cells. Furthermore, the probe AH⁺ effectively visualized pH changes in Drosophila melanogaster and zebrafish larvae, further supporting its applicability across diverse biological systems. We demonstrate that a fluorescence ratiometric intensity graph for probe AH⁺ can be effectively employed to determine pH values within the mitochondria of HeLa cells.