Fluorogenic Heterodimers Enable Rapid Fluorescence Lifetime Imaging of Prostate-Specific Membrane Antigen-Positive...

Accurate intraoperative delineation of prostate cancer margins remains a major challenge, with positive surgical margins contributing to tumor recurrence. Fluorescence-guided surgery (FGS) using near-infrared (NIR) probes targeting prostate-specific membrane antigen (PSMA) offers a promising solution but is limited by high background from "always-on" probes or slow activation kinetics of existing activatable probes. Here, we developed a modular design strategy based on fluorogenic heterodimers for rapid NIR fluorescence activation of PSMA-targeted probes. A cyanine core was conjugated with various fluorescent molecular rotors (FMRs) to generate environment-sensitive fluorophores with fast fluorescence turn-on in viscous environments. The optimal fluorophore, Cy1a, exhibited a 120-fold fluorescence enhancement and nearly 7-fold lifetime extension in vitro. Conjugation of a PSMA-targeting ligand yielded Cy2a, which rapidly activated upon PSMA binding, restoring NIR fluorescence and extending average fluorescence lifetime from 0.16 to 1.01 ns. In live cells, Cy2a enabled real-time, PSMA-specific imaging, and the fluorescence lifetime-based gating analysis allowed spatial resolution of probe-PSMA binding and dissociation. In human tissues and murine models, Cy2a achieved rapid, wash-free staining with high tumor-to-background contrast. In summary, this work establishes a versatile strategy for fast activatable NIR probes and demonstrates its potential for precise real-time imaging in prostate cancer surgery and pathology.