Reasonable Design of Near-Infrared Boron Dipyrromethene with Cationic Pyridinium for Super-Resolution Imaging of Dynamic Voltages of Mitochondria in Living Cells

Mitochondrial membrane potential (ΔΨm) is a critical regulator of cellular homeostasis and an established biomarker in mitochondrial dysfunction. While super-resolution fluorescence imaging reveals intrinsic links between mitochondrial ultrastructure and function, prolonged monitoring of the dynamic ΔΨm remains constrained by the scarcity of photostable voltage-sensitive probes. Here, we designed and synthesized three water-soluble near-infrared boron dipyrromethene (BODIPY) probes (o/m/pMePy-BDP). These cationic pyridinium-functionalized probes exhibit specific mitochondria localization (Pearson’s colocalization coefficient >0.93), high photostability (<15% intensity loss after 15 min laser irradiation), and exceptional biocompatibility. When integrated with structured illumination microscopy (SIM), they resolved mitochondrial cristae ultrastructure at 0.24 μm resolution and captured real-time ΔΨm fluctuations during fusion/fission (∼15 mV shifts) and mitochondria-lysosome contact (MLC). Semiquantitative submitochondrial models further revealed voltage gradients (150–170 mV) across cristae junctions, challenging the classical “homogeneous ΔΨm” paradigm. The probes’ compatibility with multiplexed imaging enabled continuous ΔΨm tracking during mitophagy, uncovering transient bioenergetic hotspots. This work bridges nanoscale mitochondrial dynamics to disease mechanisms, providing tools to dissect pathologies from neurodegeneration to cancer.