Real‐Time Super‐Resolution Tracking of mtDNA Remodeling and Inflammatory Release with a Selective Fluorescent Probe

Abstract Mitochondrial DNA (mtDNA) is vital for mitochondrial function and cellular homeostasis, with its spatiotemporal dynamics are tightly linked to development, metabolism, and disease progression. However, super‐resolution tracking of mtDNA in live cells remains limited by lack of selective, photostable small‐molecule probes. Here, we present mtNARed, a rationally engineered, wash‐free fluorescent probe featuring a large Stokes‐shift, and high photostability that enables super‐resolution tracking of mtDNA dynamics in live cells using stimulated emission depletion (STED) microscopy, with complementary readouts by fluorescence‐lifetime imaging microscopy (FLIM). mtNARed precisely localizes to mitochondrial nucleoids and supports long‐term imaging while minimizing interference from nuclear DNA. This capability generalizes across diverse mammalian cell types, including highly polarized sperm cells. Importantly, under mitochondrial stress or inflammatory stimulation, mtNARed reports in situ and at super‐resolution of the progressive release of mtDNA, correlating with mitochondrial depolarization, membrane disintegration, and immune activation. This work provides a robust and versatile platform for advanced mtDNA imaging, opening opportunities to dissect mitochondrial genome dynamics, maintenance, and signaling across physiological and pathological states.