Optimization of the optical transparency of bones by PACT-based passive tissue clearing
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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.
Abstract
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.
참고문헌 (39)
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