Real-Time Monitoring of Mitochondrial pH in HeLa Cells and Drosophila melanogaster Larvae Using BODIPY-Based...

Three BODIPY-based fluorescent probes, AH⁺, BH⁺, and CH⁺, were synthesized for ratiometric pH sensing in living cells and fruit fly larvae. These probes were designed to target mitochondrial environments by functionalizing the BODIPY core with various substituent groups that tune their 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 a broad-range pH monitoring across a wide pH spectrum (3.5 to 9.1). These ratiometric pH sensors display good reversibility and response time under varying pH conditions. In application, the probe AH⁺ was utilized to monitor pH fluctuations under conditions of oxidative stress and starvation conditions. Dual-channel cell imaging revealed a pH-dependent fluorescence shift with precise transitions, demonstrating the feasibility of real-time monitoring of mitochondrial pH changes in living cells. Furthermore, the probe AH⁺ effectively visualized pH changes in Drosophila melanogaster 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.