Transient optical clearing using absorbing molecules for ex-vivo and in-vivo imaging

Abstract

Dynamic imaging is a foundational tool in biology and medicine but is limited by light scattering in live tissues caused by refractive index mismatches, which reduces penetration depth and resolution in biological tissues. Our previous work introduced a novel approach to achieve an optical transparency in live animals by utilizing strongly absorbing molecules. When these absorbing molecules, such as Tartrazine, an FDA approved dye commonly used in foods, dissolve in water, they modify the refractive index of the aqueous medium through Kramers-Kronig relations. This modulation allows reduction of the refractive index mismatch between separate components in tissue with distinct refractive indices, thus mitigating scattering. In this article, we report detailed methodology of our tissue clearing technique using a Tartrazine-based aqueous solution, applied to both ex-vivo and in-vivo samples. This approach reversibly renders live mice bodies transparent in the visible spectrum, enabling clearer and deeper imaging of internal processes and structures. We then utilized an advanced image processing algorithm to improve the contrast and segmentation of organs of interests. We also show high transparency in ex-vivo samples, such as chicken breast, when treated with Tartrazine based solution. This method offers a versatile and accessible approach to enhancing imaging capabilities across various modalities and paves the way for advancement in our understanding of complex biological systems. SUMMARYOptical clearing techniques, such as absorbing molecules solutions, reduce light scattering temporarily to enhance biological imaging and offer biocompatible, reversible in-vivo imaging strategies for deeply embedded organs. This protocol presents detailed experimental procedures as well as advanced image processing to achieve high-quality, dynamic imaging for real-time dynamics in living animals.