A rapid and universal method for depth estimation of lesions in heterogeneous tissues via photosafe ratiometric transmission Raman spectroscopy

Noninvasive detection of deep lesions remains a long-standing goal for clinical applications, with depth estimation of a single lesion in heterogeneous tissues being a key challenge. Currently, optical techniques are widely applied for lesion detection, but they face difficulties in achieving rapid and precise lesion depth estimation, particularly when the lesions are buried in thick heterogeneous tissues and the applied irradiance is below clinically maximum permissible exposure. Herein, we theoretically and experimentally demonstrate a universal method for depth prediction of phantom lesions labeled with surface-enhanced Raman scattering nanotags in thick biological tissues using ratiometric transmission Raman spectroscopy (TRS). We begin by highlighting the linear relationship between the natural logarithm of Raman peak-to-peak ratio and the lesion depth and establishing a home-built TRS system with the clinically safe irradiance. We achieve an accurate depth prediction for phantom lesions hidden in 6-cm-thick ex vivo homogeneous tissue with a root mean squared error (RMSE) as low as 2.42%. Additionally, we predict the depth of phantom lesions buried in 5-cm-thick ex vivo heterogeneous tissues with an RMSE of down to 8.35%. We also demonstrate the applicability of this method theoretically for highly heterogeneous tissues such as complex in vivo environments. This work provides a rapid, robust, and universal method to estimate the depth of lesions in complex biological samples, demonstrating the potential of ratiometric Raman spectroscopy for lesion localization in clinical applications.