High Spatiotemporal Near‐Infrared II Fluorescence Lifetime Imaging for Quantitative Detection of Clinical Tumor Margins

Accurate detection of tumor margins is essential for successful cancer surgery. While indocyanine green (ICG)-based near-infrared (NIR) fluorescence (FL) surgical navigation enhances the visual identification of tumor margins, its accuracy remains subjective, underscoring the need for quantitative approaches. In this study, a high spatiotemporal fluorescence lifetime (FLT) imaging technology is developed in the second near-infrared window (NIR-II, 1000-1700 nm) for quantitative tumor margin detection, utilizing folate receptor-targeted ICG nanoprobes (FPH-ICG). FPH-ICG exhibits a significantly prolonged NIR-II FLT (750 ± 7 ps vs 260 ± 3 ps) and increased NIR-II FL brightness (FPH-ICG/ICG = 3.8). In vitro and in vivo studies confirm that FPH-ICG specifically targets folate receptor-α (FRα) on SK-OV-3 ovarian cancer cells, achieving high-contrast NIR-II FL imaging with a signal-to-background ratio of 10.8. Notably, NIR-II FLT imaging demonstrates superior accuracy (90%) and consistency in defining tumor margins compared to NIR-II FL imaging (58%) in both SK-OV-3 tumor-bearing mice and clinical tumor samples. These findings underscore the potential of NIR-II FLT imaging as a quantitative tool for guiding surgical tumor margin detection.