Facilitating Quantitation of Mitochondrial G-Quadruplex DNA with an Iridium(III) Two-Photon Phosphorescence Lifetime Imaging Probe

G-quadruplex (G4) DNA is a critical target for disease monitoring. However, while existing probes primarily focus on nuclear G4s, their mitochondrial counterparts (mitoG4) remain underexplored. The few available mitoG4 probes are limited by the shallow penetration depth of one-photon excitation and generally permit only qualitative detection. To address these challenges, we developed a mitochondria-localized iridium(III) complex, Ir2PDP. By screening a library of seventy-five distinct mitoG4 sequences, we validated its specificity, which results in up to a 113.4-fold emission enhancement and a 740 ns lifetime extension, all without perturbing native mitoG4 dynamics. Owing to its large two-photon absorption cross-section and exceptional antiphotobleaching capability, Ir2PDP enables two-photon phosphorescence lifetime imaging (PLIM) that differentiates eight specific mitoG4 structures and quantifies their abundance in live cells and zebrafish. To the best of our knowledge, Ir2PDP represents the first two-photon PLIM probe capable of quantitative mitoG4 DNA detection.