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 (ΔΨ m ) 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 ΔΨ m 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/p MePy-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 ΔΨ m 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 ΔΨ m ” paradigm. The probes’ compatibility with multiplexed imaging enabled continuous ΔΨ m 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.