Enhancing the efficiency of achieving optical transparency in live animals using absorbing molecules

In vivo optical imaging is crucial for studying disease mechanisms but is limited by light scattering and poor penetration in biological tissues. While tissue-clearing reagents (hydrophilic/hydrophobic) and bioluminescent probes improve imaging, achieving effective optical transparency in live tissues remains a challenge. This study builds on recent work using absorbing dyes (tartrazine and 4-aminoantipyrine) to enhance in vivo tissue clearing, aiming to optimize efficacy and biosafety.

We aimed to develop a mixed solution of tartrazine and 4-aminoantipyrine (4-AA) that improves optical transparency, accelerates clearing, and reduces toxicity compared to individual dyes, enabling safer and more efficient deep-tissue imaging in live animals.

The study employed a multi-pronged experimental approach: solution optimization involved testing varying ratios of tartrazine and 4-AA (5:1, 10:1) to characterize their optical properties through UV-Vis-NIR spectroscopy and refractive-index measurements, while simultaneously evaluating ex vivo skin-clearing efficacy; in vivo validation was conducted by applying the optimized gels to depilated mouse skin and systematically recording key parameters including transparency-onset time, maximum clearing duration, and light transmittance; concurrent biosafety assessments monitored critical health indicators such as animal survival rates, longitudinal weight changes, and liver/kidney function markers [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA)] during the post-treatment period.

The optimized mixed solutions (5:1 and 10:1 tartrazine:4-AA ratios) demonstrated superior clearing efficiency, achieving faster tissue transparency than tartrazine alone while matching the performance of 4-AA but with significantly reduced toxicity. Optical characterization revealed stable refractive indices ( $\sim 1.42$ ) and strong absorption across visible/NIR wavelengths for all formulations. While 4-AA alone exhibited severe hepatorenal toxicity and 100% mortality (3/3 mice), the 5:1 mixed solution maintained efficacy with no mortality and only mild ALT/AST elevation. Transmittance measurements showed 4-AA gels achieved $\sim 40\%$ light transmission, whereas mixed gels reached $\sim 15\%$ due to tartrazine's residual absorption in the red-NIR spectrum, suggesting an optimal balance between clearing performance and biosafety in the composite formulations.

The 5:1 tartrazine:4-AA cocktail optimally balances speed, clarity, and biosafety, advancing in vivo tissue-clearing technology. This strategy addresses key limitations of stand-alone dyes and expands potential applications in biomedical imaging, such as 3D tumor visualization and dynamic pathology studies. Future work should refine ratios for diverse tissues and integrate auxiliary agents (e.g., surfactants) to further enhance clearing.