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.

Figure1 Schematic illustration of the non-invasive detection and depth estimation of in vivo deep sentinel lymph node (SLN) on a rat model using TRS. The SERS NPs are injected on the foot pad and migrate to the SLN, which can then be non-invasively identified from the unique Raman signal of SERS NPs using the TRS setup. Based on the Raman signal peak ratio, the depth of in vivo SLN can be determined. The location of SLN can be obtained by TRS mapping. This pre-operative location of SLN enables the accurate removal of SLNs with minimal invasion.

Figure 2 Preparation and characterization of SERS NPs. a) Scheme of SERS NP preparation. b) TEM images, c) UV–vis spectra, d) zeta potential, and e) hydrodynamic size of i) Au core NPs, ii) Au@Ag GERRTs, and iii) GERRTs with PEG layer modification. f) Raman spectra of PEG-modified GERRTs with different concentrations. All Raman measurements were performed at the wavelength of 785 nm with 10 mW laser power and 10 ms integration time. Data were repeatedly measured three times and mean ± SD waspresented.

Figure 3 Non-invasive in vivo detection and depth prediction of the rat popliteal SLN using SETRS within MPE. a) Schematic illustration of SERS NPs injection into the foot pad and migration to SLN. b) TRS setup and c) measurements on a rat leg to locate the lymph node. d) TRS measurements on the rat leg and the spectra collected at the corresponding marker position (n = 3). The laser irradiance was 0.105 J cm−2 and integration time was 0.5 s. e) Backscattering Raman measurements on the rat leg and corresponding spectra (n = 3). The laser irradiance is 1.31 J cm−2 and integration time was 10 ms. The bars in the bright-field images are 1 cm for (d) and (e). Both scanning areas were of 18 × 10 mm2 (10 × 6 pixels) and the measurements were repeated three times for each point. f) Schematic diagram of depth prediction for SLN. g) Predicted depth and h) experimental depth of the SLN, which was stained with methyl blue dye (n = 3).

DOI: 10.1002/advs.202301721