Tissue clearing
Tissue clearing refers to a group of chemical techniques used to turn tissues transparent. By turning tissues transparent to certain wavelengths of light, it allows one to gain optical access to a tissue. That is, light can pass into and out of the cleared tissue freely, allowing one to see the structures deep within the tissue without physically cutting it open. Many tissue clearing methods exist, each with different strengths and weaknesses. Some are generally applicable, while others are designed for specific applications. Tissue clearing is usually useful only combined with one or more fluorescent labeling techniques such as immunolabeling and subsequently imaged, most often by optical sectioning microscopy techniques. Tissue clearing has been applied to many areas in biological research. It is one of the more efficient ways to perform three-dimensional histology.
History
In the early 1900s, Werner Spalteholz developed a technique that allowed the clarification of large tissues, using Wintergrünöl and benzyl benzoate. Various scientists then introduced their own variations on Spalteholz's technique. Tuchin et al. introduced tissue optical clearing in 1997, adding a new branch of tissue clearing that was hydrophilic instead of hydrophobic like Spalteholz's technique. In the 1980s, Andrew Murray & Marc Kirschner developed a two-step process, wherein tissues were first dehydrated with alcohol and subsequently made transparent by immersion in a mixture of benzyl alcohol and benzyl benzoate, a technique they coupled with light sheet fluorescence microscopy, which remains the method with the highest clearing efficacy to date, regardless any tissue pre-processing step. In the most extreme case, it allows the clearing of a whole mouse of even a whole human brain.In 2024, Hong, Brongersma, and Ou reported that applying high concentrations of the food dye tartrazine could transiently and reversibly increase the optical transparency of certain biological tissues, including the skin, in live mice. The authors attributed this effect to tartrazine's strong absorption in the blue region of the visible spectrum and to refractive index modulation at longer wavelengths, consistent with the Kramers–Kronig relations. Following publication, the findings have been independently reproduced and extended by multiple laboratories in several subsequent studies. Specifically, this in vivo optical clearing approach has been applied by multiple independent laboratories to enhance imaging depth in modalities such as optical coherence tomography and photoacoustic imaging. In 2025, Valery V. Tuchin, a pioneer in hydrophilic tissue clearing, demonstrated tartrazine can make the skull more transparent in live mice, enabling transcranial laser speckle imaging of cortical blood flow in real time. In addition, a number of other labs have demonstrated the utility of tartrazine to enable deep-tissue Raman sensing and fluorescence lifetime imaging. In addition to tartrazine, several other absorbing dye molecules, including the FDA-approved contrast agents fluorescein and indocyanine green, have also been repurposed to function as in vivo optical clearing agents. This observation suggests that the underlying physical principle of dye-enabled optical clearing is not limited to a single molecule and that multiple dye molecules may be repurposed as tissue clearing agents.