Reaction-free and MMP-independent fluorescent probes for long-term mitochondria visualization and tracking

Visualizing and tracking mitochondrial dynamic changes is crucially important in the fields of physiology, pathology and pharmacology. Traditional electrostatic-attraction based mitochondrial probes fail to visualize and track the changes due to their leakage from mitochondria when mitochondrial membrane potential (MMP) decreases. Reaction-based MitoTracker probes can realize visualization and tracking of mitochondria changes independent of MMP changes. However, such probes impair mitochondrial proteins and exhibit high cytotoxicity. Therefore, it still remains challenging to explore reaction-free and highly biocompatible probes for visualizing and tracking mitochondrial dynamics independent of MMP fluctuations. Herein we synthesized two reaction-free fluorescent mitochondrial probes ECPI-12 and IVPI-12 bearing a long C₁₂-alkyl chain. These cationic probes can firmly immobilize in the mitochondrial inner membrane by strong hydrophobic interaction between the C₁₂-alkyl chain and lipid bilayer, resulting in high specificity and long-term mitochondrial staining regardless of MMP changes. They also exhibit large two-photon absorption cross-sections and show deep penetration in live tissues in two-photon microscopy. Furthermore, they display excellent biocompatibility and realize <i>in situ</i> and real-time mitophagy tracking in live cells. These excellent properties could make ECPI-12 and IVPI-12 the first selective tools for long-term visualization and tracking of mitochondrial dynamics.