Ratiometric Near-Infrared Fluorescence Probes for Quantitative Monitoring of Mitochondrial pH Dynamics in Drosophila Melanogaster and HeLa Cells during Metformin Treatment

Abstract

Starvation Hypoxia Oxidative stressFly larvaeHighlights• Two ratiometric fluorescent probes, AH⁺ and BH⁺, were synthesized from IR-780 iodide, incorporating imidazole, hydroxy-xanthene, and hemicyanine groups for selective mitochondrial targeting.• The probes operate via deprotonation-induced π-conjugation rearrangements, exhibiting dual-emission fluorescence with large Stokes shifts (>90 nm) in their protonated forms and anti-Stokes shifts in their unprotonated forms.• High photostability, selectivity, reversibility, and low cytotoxicity were confirmed in HeLa cells, with strong mitochondrial colocalization (PCC = 0.96 for AH⁺, 0.93 for BH⁺). The probes successfully monitored pH changes under diverse stress conditions and in Drosophila melanogaster larvae.• Metformin treatment was shown to alter mitochondrial pH, underscoring the utility of AH⁺ and BH⁺ in studying drug effects and establishing them as valuable tools for investigating mitochondrial function and cellular stress responses in health and disease.