Non‐Invasive Detection, Precise Localization, and Perioperative Navigation of In Vivo Deep Lesions Using Transmission Raman Spectroscopy

Abstract Non‐invasive detection and precise localization of deep lesions have attracted significant attention for both fundamental and clinical studies. Optical modality techniques are promising with high sensitivity and molecular specificity, but are limited by shallow tissue penetration and the failure to accurately determine lesion depth. Here the authors report in vivo ratiometric surface‐enhanced transmission Raman spectroscopy (SETRS) for non‐invasive localization and perioperative surgery navigation of deep sentinel lymph nodes in live rats. The SETRS system uses ultrabright surface‐enhanced Raman spectroscopy (SERS) nanoparticles with a low detection limit of 10 pM and a home‐built photosafe transmission Raman spectroscopy setup. The ratiometric SETRS strategy is proposed based on the ratio of multiple Raman spectral peaks for obtaining lesion depth. Via this strategy, the depth of the phantom lesions in ex vivo rat tissues is precisely determined with a mean‐absolute‐percentage‐error of 11.8%, and the accurate localization of a 6‐mm‐deep rat popliteal lymph node is achieved. The feasibility of ratiometric SETRS allows the successful perioperative navigation of in vivo lymph node biopsy surgery in live rats under clinically safe laser irradiance. This study represents a significant step toward the clinical translation of TRS techniques, providing new insights for the design and implementation of in vivo SERS applications.