Alkyl chain elongation engineering: Unlocking high‐brightness NIR‐II O‐xanthene dyes for in vivo bioimaging

Abstract NIR‐II fluorescent probes are powerful tools for advanced imaging, with performance governed by molar extinction coefficient (ε), quantum yield (Φ), and brightness (ε × Φ). However, achieving high brightness remains a major challenge. Here we report SCU‐OX‐Hex, a D–A–D probe with an O‐xanthene acceptor and an N‐alkylated aniline donor. Elongating the N‐alkyl chain enhances Φ and brightness while suppressing aggregation‐caused quenching. SCU‐OX‐Hex exhibits ε = 1.71 × 10 5 M −1 cm −1 , Φ = 0.57%, and a brightness of 974.7 M −1 cm −1 —among the highest reported for organic NIR‐II probes. In vivo, it enables high‐resolution imaging of the abdominal vasculature, the gastrointestinal tract, and tumor angiogenesis. In a lymph node metastasis model, it provides superior contrast and resolution compared with ICG, enabling precise intraoperative identification. This work establishes SCU‐OX‐Hex as a high‐performance NIR‐II agent and offers a broadly applicable design strategy for next‐generation probes.