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Intraoperative near-infrared II (NIR-II) fluorescence imaging offers high-contrast, real-time visualization during surgery, yet the development of activatable probes remains a formidable challenge because most tissue-specific biomarkers, such as receptor proteins, are chemically inert intrinsic. Here, we present a protein-triggered reassembly strategy in which planar NIR-II dyes pre-assemble into π-π stacked aggregates with quenched fluorescence that reorganize into highly emissive dye-protein complexes upon target binding, thereby achieving activatable intraoperative imaging. Compared with indocyanine green (ICG), a clinically approved "always-on" cholangiography agent, quinocyanine green (QCG) was developed as a bile specifical activated NIR-II probe through stepwise molecular engineering. Native PAGE, protein sequencing, and TEM analyses all revealed that a hemoglobin subunit in bile drives the transformation of the initial QCG nanosheets into uniform homogeneous spherical protein-dye complexes, yielding a remarkable 310-fold fluorescence enhancement at 1011 nm. Taking such advantage, for the first time, we have clearly and directly visualized intrahepatic bile duct in vivo. Moreover, QCG afforded high-contrast, precise intraoperative cholangiography, allowing detailed biliary tree mapping, stricture identification, bile leakage detection, and dual-color hepatobiliary imaging. This in situ reassembly strategy establishes a generalizable molecular design principle for activatable NIR-II probes and expands the toolbox for precision fluorescence-guided surgery.
Zhang et al. (Mon,) studied this question.