Spectrally Silent and Optically Transparent: Clear-SiR for Deep Raman Biomolecular Sensing.

Abstract

Raman spectroscopy (RS) offers highly specific, label-free molecular identification, yet its application in deep-tissue imaging is fundamentally limited by light scattering and spectral interference from endogenous biomolecules. A promising strategy to mitigate these issues involves using vibrational tags that emit signals within the Raman-silent region (1800-2800 cm-1), minimizing background noise. However, these signals remain susceptible to attenuation due to the inherent optical opacity of biological tissues caused by refractive index (RI) mismatches among tissue constituents. Here, we introduce Clearing-enabled Light Enhancement for Analytical Raman in the Silent Region (Clear-SiR), a hybrid method that combines RI modulation through tissue clearing with spectral targeting in the silent region to enable high-fidelity Raman detection through biological tissue. Utilizing tartrazine, an FDA-approved dye with strong short-wavelength absorption, Clear-SiR homogenizes tissue RI based on the Kramers-Kronig relations, enhancing optical transparency and reducing light scattering. Paired with 4-aminobenzonitrile (4-ABN) as a nitrile-based silent region reporter, we demonstrate that Clear-SiR achieves up to a nearly 2-fold increase in detection depth under red (638 nm) and near-infrared (785 nm) excitation. We further use a rapid, gel-based tartrazine delivery protocol suitable for clinical and point-of-care settings. Our proof-of-concept results demonstrate that Clear-SiR overcomes both spectral and optical barriers for transcutaneous measurement, enabling highly accurate, deep-tissue biochemical sensing with minimal distraction from endogenous signals. By integration of physical tissue clearing with spectral targeting in the Raman-silent region, this method establishes a robust platform for probing molecular architecture at clinically relevant depths. Clear-SiR opens new avenues for noninvasive molecular phenotyping and longitudinal monitoring of pathophysiological process in organoids and intact tissues.