Dual‑Channel Hypoxia Detection Using PET‐Based Water‐Soluble Cyanine Probes to Suppress False Negatives in Image‑Guided Pancreatic Cancer Resection

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.