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

Super-resolution live-cell imaging demands probes with sustained photostability, precise subcellular localization, and minimal cytotoxicity. Commercial probes such as MitoTracker photobleach rapidly, retaining only ~14% of initial fluorescence after 40 s of continuous 488 nm irradiation. Polymeric AIEgens offer superior photostability via restriction of intramolecular motion (RIM), yet their pre-assembled structures are prone to lysosomal entrapment, precluding broader subcellular accessibility.

Herein, we present Organelle Localization-Induced Bio-orthogonal Polymerization (OLIBOP), which resolves this delivery-stability paradox by synthesizing the polymer directly within the target organelle. The small-molecule precursor 1-AIE passively diffuses into cells and undergoes GSH-triggered CBT–Cys polymerization specifically within mitochondria, exploiting the mitochondrial membrane potential (ΔΨm = −150 to −180 mV) for an estimated >1000-fold local accumulation. The resultant poly-AIEgen (Mw = 29–44 kDa) retains >40% fluorescence after 40 s of irradiation, maintains robust signal over 48 h post-washout, and exhibits >80% cell viability up to 40 µM.

Phasor-FLIM tracked the progressive lifetime elongation accompanying polymerization over 24 h in live cells, affording direct spatiotemporal visualization of intracellular polymer formation. Airyscan microscopy enabled unambiguous imaging of mitochondrial cristae and real-time tracking of fusion and fission dynamics. Upon substitution of the targeting moiety with cyclo(RGD), OLIBOP was redirected to lysosomes, affording pH-resistant long-term lysosomal imaging (Pearson's r = 0.93 vs. LysoTracker).

Collectively, OLIBOP establishes a generalizable platform for intracellular construction of organelle-specific photostable probes, with direct implications for long-term super-resolution interrogation of organelle dynamics.