In Vivo Surface-Enhanced Transmission Raman Spectroscopy and Impact of Frozen Biological Tissues on Lesion Depth Prediction

Plasmonic surface-enhanced transmission Raman spectroscopy (SETRS) has emerged as a promising optical technique for detecting and predicting the depths of deep-seated lesions in biological tissues. However, in vivo studies using SETRS are scarce and typically show shallow penetration depths. Moreover, the optical parameters used in the prediction process are often derived from frozen samples and there is limited understanding of how freezing affects the optical properties of biological tissues and the accuracy of depth prediction in living models. In this work, we conduct in vivo SETRS measurements on thick abdominal tissue region of the live rats to investigate the impact of freezing on the measured optical properties for the purpose of depth prediction. First, we fabricated ultrahigh bright surface-enhanced Raman spectroscopy (SERS) nanotags and utilized a custom transmission Raman system. We then measured the change of optical attenuation at two different wavelengths (Δμ) for four types of rat tissues (including skin, fat, muscle, and liver) following freezing. The freezing process dramatically affects Δμ values, even after only 1 day of freezing. In contrast, Δμ values obtained from fresh samples enable precise localization of SERS lesion phantoms in the live rat with only 5% deviation. The total thickness of the live rat is 2.6 cm, which, to the best of our knowledge, is the highest value of in vivo SETRS studies so far. This work helps to fill the gap in the SERS field of tissue localization and optical coefficient studies in highly heterogeneous tissues, and demonstrates the potential of the SETRS technique to achieve precise clinical localization of deep lesions.