Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, United States.
Division of Cardiothoracic Surgery, Spectrum Health, Grand Rapids, MI, United States; Department of Cardiac Surgery, Medical University of Silesia, School of Medicine in Katowice, Katowice, Poland.
J Biomech. 2021 Jun 9;122:110413. doi: 10.1016/j.jbiomech.2021.110413. Epub 2021 Apr 5.
Optical clearing of biological tissues improves imaging depth for light transmission imaging modalities such as two-photon microscopy. In studies that investigate the interplay between microstructure and tissue-level mechanics, mechanical testing of cleared tissue may be useful. However, the effects of optical clearing on soft tissue mechanics have not been investigated. Thus, we set out to quantify the effects of a simple and effective optical clearing protocol on the mechanics of soft collagenous tissues using ovine mitral valve anterior leaflets as a model system. First, we demonstrate the effectiveness of an isotonic glycerol-DMSO optical clearing protocol in two-photon microscopy. Second, we evaluate the mechanical effects of optical clearing on leaflets under equibiaxial tension in a dependent study design. Lastly, we quantify the shrinkage strain while traction-free and the contractile forces while constrained during clearing. We found the optical clearing protocol to improve two-photon imaging depth from ~100 μm to ~500-800 μm, enabling full-thickness visualization of second-harmonic generation, autofluorescent, and fluorophore-tagged structures. Under equibiaxial tension, cleared tissues exhibited reduced circumferential (p < 0.001) and radial (p = 0.009) transition stretches (i.e. stretch where collagen is recruited), and reduced radial stiffness (p = 0.031). Finally, during clearing we observed ~10-15% circumferential and radial compressive strains, and when constrained, ~2mN of circumferential and radial traction forces. In summary, we suggest the use of this optical clearing agent with mechanical testing be done with care, as it appears to alter the tissue's stress-free configuration and stiffness, likely due to tissue dehydration.
生物组织的光学透明化可以提高光传输成像模式（如双光子显微镜）的成像深度。在研究微结构与组织水平力学之间相互作用的研究中，对透明化组织进行力学测试可能是有用的。然而，光学透明化对软组织力学的影响尚未得到研究。因此，我们旨在使用绵羊二尖瓣前瓣作为模型系统，定量研究一种简单有效的光学透明化方案对软胶原蛋白组织力学的影响。首先，我们证明了等渗甘油-DMSO 光学透明化方案在双光子显微镜中的有效性。其次，我们在一项依赖研究设计中评估了光学透明化对叶瓣在等张拉伸下的力学影响。最后，我们量化了在无张力状态下的收缩应变和在透明化过程中受到约束时的收缩力。我们发现，光学透明化方案将双光子成像深度从约 100μm 提高到约 500-800μm，从而能够对二次谐波产生、自发荧光和荧光标记结构进行全厚度可视化。在等张拉伸下，透明化组织的周向（p<0.001）和径向（p=0.009）过渡拉伸（即胶原蛋白募集时的拉伸）以及径向刚度（p=0.031）降低。最后，在透明化过程中，我们观察到约 10-15%的周向和径向压缩应变，以及当受到约束时，约 2mN 的周向和径向牵引力。总之，我们建议在进行光学透明化与力学测试时要小心使用这种透明化剂，因为它似乎会改变组织的无应力构型和刚度，这可能是由于组织脱水所致。
