Multimodal Imaging of Live Cells and Tumor Spheroids by a G‐Quadruplex DNA Selective Ru(II)Polypyridyl Probe

G-quadruplex (G4) DNA structures are emerging therapeutic-targets across numerous disease states. Consequently, highly selective, small-molecule luminescent probes are required to monitor G4 prevalence, dynamics, and therapeutic responses in live-cells. Although ruthenium complexes have been widely explored as multimodal DNA and G4 probes in solution, a G4-selective ruthenium probe capable of functioning inside live cells has remained elusive. Here, we report the application of a G4-selective, luminescent "light-switch" ruthenium(II) complex, Ru-PDC3, as a selective probe in cellulo. While Ru-PDC3 was previously known to bind G4 with high affinity and induce solution-phase destabilization, we demonstrate that it preferentially localizes within the nuclei of live HeLa cells, enabling the dynamic tracking of G4 structures. The G4-selective binding in cellulo was confirmed via phosphorescence lifetime imaging microscopy, Raman mapping, enzymatic digestion, and colocalization studies with the G4-specific antibody, BG4. Furthermore, increasing the complex concentration or visible light irradiation intensity beyond standard imaging conditions induced phototoxicity. Mechanistic investigations employing in cellulo reactive oxygen species (ROS) scavengers and plasmid-DNA cleavage assays confirmed that phototoxicity is derived from singlet oxygen generation with a contribution from radical species. Together, these findings establish Ru-PDC3 as a powerful dual-functional tool for both the real-time visualization and potentially targeted photodynamic therapy of G4-DNA.