Accurate intraoperative delineation of tumor margins remains a major clinical challenge because conventional fluorescence imaging is strongly influenced by probe concentration, tissue heterogeneity, and imaging conditions. Here, we report a renally clearable, tumor-targeted molecular probe, NY-07, for quantitative tumor-margin delineation using second near-infrared (NIR-II) fluorescence lifetime (FLT) imaging. NY-07 is constructed by conjugating a cyanine-based fluorophore with pemetrexed as an active targeting ligand, yielding a clinically compatible probe with excellent solubility, high optical stability, and efficient renal clearance. NY-07 exhibits tail emission in the NIR-II window with a stable fluorescence lifetime of 480 ± 0.6 ps and NIR-II brightness approximately twice that of indocyanine green (ICG) at the same concentration, while retaining ∼70% of its NIR-II signal after 7 days of storage. In vivo imaging demonstrates rapid renal excretion, with strong kidney and bladder signals within seconds after injection and minimal hepatic accumulation. Using cell models, tumor-bearing mice, and patient-derived tissues, we show that NY-07 enables target-specific NIR-II FLT imaging and achieves quantitative discrimination between tumor, inflammation, and normal tissues based on distinct lifetime signatures, enabling precise visualization of tumor margins independent of probe concentration and imaging parameters. Compared with conventional NIR-II fluorescence intensity (FLI) imaging, FLT imaging significantly reduces false-positive signals and improves boundary definition in complex biological environments. This work establishes a clinically translatable strategy that integrates renal clearance, active tumor targeting, and lifetime-resolved imaging for functional tumor-margin delineation, providing a robust platform for improving surgical precision and advancing next-generation image-guided oncologic surgery.
Zhang et al. (2026) studied this question.