Organelle Localization‐Induced Bio‐Orthogonal Polymerization (OLIBOP) for Photostable Super‐Resolution Live‐Cell Imaging

Real-time monitoring of dynamic biological processes demands fluorescent probes that can withstand prolonged light exposure without photobleaching-a critical limitation that to long-term live-cell imaging studies. Polymeric AIEgens possessed superior photostability with biocompatibility, making them attractive for bioimaging applications. However, their assembled structures often bias localization toward lysosomes, underscoring the need for probes with broader subcellular accessibility. Herein, we resolve this paradox through Organelle Localization-Induced Bio-orthogonal Polymerization (OLIBOP), a paradigm-shifting approach that delivers small molecules to specific organelles where they are synthesized into photostable polymeric fluorescent probes in situ. The designed small-molecule precursor, 1-AIE, incorporates triphenylamine for AIE effect, pyridinium for mitochondria-targeting moiety, and CBT-Cys moiety for bio-orthogonal condensation by a GSH-responsive disulfide trigger. Upon cellular uptake, 1-AIE undergoes reduction-triggered polymerization specifically at mitochondrial sites, transforming from a deliverable monomer into a photostable polymeric probe. Using phasor-FLIM analysis, we visualized this remarkable in situ transformation and demonstrated that the resulting poly-AIEgen exhibits dramatically enhanced fluorescence intensity and extended lifetime. Most importantly, the exceptional photostability of these in situ-formed probes enabled unprecedented real-time tracking of mitochondrial dynamics over extended periods. OLIBOP represents a conceptual breakthrough that overcomes the delivery-stability trade-off of polymeric AIEgnes, opening new possibilities for high-resolution, long-term live-cell imaging with superior biocompatibility.