Ferroptosis‐Impelled NIR Cyanine Dyes in Situ Generation Enabled Zero‐Background Fluorescence Imaging and Precise Surgical Tumor Excision

Abstract The diagnostic accuracy of fluorescence imaging enabled lesion site visualization and pathological biomarker quantification is often compromised by non‐specific probe activation and “high‐background” fluorescence interference, which contribute to false‐positive signals and reduced signal‐to‐noise ratio, ultimately limiting clinical reliability. Herein, we propose a “zero‐background” fluorescence imaging strategy for precise quantification and evaluation of hydroxyl radical (·OH) levels in sorafenib‐provoked ferroptotic tumor. This study utilizes endogenous ·OH generated during tumorous ferroptosis as biocatalyst, triggering the heterogenous dimerization of monomers for directly in‐situ formulating near‐infrared (NIR) emissive cyanine dyes. The octadecyl‐substituted monomer (Cy420) demonstrated morphological variations from orbicular to square nano‐assemblies modulated with serum albumin and realized the targeted and prolonged retention in tumors facilitated with the enhanced permeability and retention (EPR) effect. Crucially, the in situ generated NIR dye (mCy849) forms a shielding effect via hydrophobic bis‐octadecyl chain‐driven self‐assembly, thus resisting molecular oxidation and ensuring quantification accuracy. In vivo experiments further demonstrated that Cy420 enables accurate zero‐background NIR imaging in sorafenib‐administrated xenograft hepatic tumor, with significantly enhanced tumor‐to‐background ratio (TBR 13.2 ± 0.74) at 72 h, competently empowered NIR fluorescence‐navigated precise tumor resection. Our strategy has provided a new paradigm for chemotherapy‐imaging synergistic regulation of tumors with authentic “zero‐background” fluorescence.