Optimization of the optical transparency of bones by PACT-based passive tissue clearing

Recent developments in tissue clearing methods such as the passive clearing technique (PACT) have allowed three-dimensional analysis of biological structures in whole, intact tissues, thereby providing a greater understanding of spatial relationships and biological circuits. Nonetheless, the issues that remain in maintaining structural integrity and preventing tissue expansion/shrinkage with rapid clearing still inhibit the wide application of these techniques in hard bone tissues, such as femurs and tibias. Here, we present an optimized PACT-based bone-clearing method, Bone-mPACT+, that protects biological structures. Bone-mPACT+ and four different decalcifying procedures were tested for their ability to improve bone tissue clearing efficiency without sacrificing optical transparency; they rendered nearly all types of bone tissues transparent. Both mouse and rat bones were nearly transparent after the clearing process. We also present a further modification, the Bone-mPACT+ Advance protocol, which is specifically optimized for processing the largest and hardest rat bones for easy clearing and imaging using established tissue clearing methods. An optimized imaging technique specifically designed for large, hard bones could significantly enhance evaluations of animal models of different bone diseases. To fully visualize and understand the structure of complex biological tissues, scientists use techniques known as 'optical clearing' to enhance the resolution and optical clarity of images. One such technique is passive clearing (PACT), which has already produced high-quality, three-dimensional analysis of multiple biological tissues. However, using PACT for large, hard bone samples is challenging and time-consuming. Now, Yong Eun Cho at Yonsei University in Seoul, South Korea, and co-workers have developed an optimized method for bone clearing called Bone-mPACT+. Their technique, which the team trialed on rat femurs and tibias, dramatically improved clearing efficiency for dense bones without losing optical transparency and image resolution.

Automated summary

Recent developments in tissue clearing methods allow three-dimensional analysis of biological structures, providing a greater understanding of spatial relationships. However, maintaining structural integrity and preventing tissue expansion/shrinkage in hard bone tissues remains a challenge.