Dual‑Channel Hypoxia Detection Using PET-Based Water-Soluble Cyanine Probes to Suppress False Negatives in...

Fluorescence‑guided surgery using activatable probes is promising, but single‑channel probes often give false negatives-especially in pancreatic cancer due to tumor heterogeneity and limited imaging depth. Here, we built a universal charge-switching gated PET platform based on cyanine dyes bearing a meso-quinolinium moiety. This platform-by decoupling quenching from the recognition group-enabled the construction of two structurally analogous, hypoxia-activatable near-infrared (NIR) probes (Cy5-Ql-NTR and Cy7-Ql-NTR), which have comparable responsive kinetics (sensitive, fast-responding, and low-background), good water solubility and favorable renal clearance, but differ in tissue imaging depth: the former generates brighter signals in superficial regions, while the latter enables deeper tissue penetration and serves as a self-calibration reference. Notably, compared to a single probe, the combined metabolic distribution of the two probes covers a broader range of tumor target tissues. Through their dual-channel imaging, depth-dependent signal attenuation is corrected, and blind spots during probe delivery are reduced. Compared to single-channel fluorescence-guided surgery in subcutaneous pancreatic cancer and orthotopic pancreatic tumor, the dual-channel approach yielded clean margins, preserved significantly more normal tissue, and left no residual tumor on histology. This self-referenced detection strategy suppresses false negatives in single-channel detection, offering a practical and optimized solution for intraoperative guidance in pancreatic cancer.