PDF(Pubmed)
Abstract：
Studies of morphology and developmental patterning in adult stages of many invertebrates are hindered by opaque structures, such as shells, skeletal elements, and pigment granules that block or refract light and necessitate sectioning for observation of internal features. An inherent challenge in studies relying on surgical approaches is that cutting tissue is semi-destructive, and delicate structures, such as axonal processes within neural networks, are computationally challenging to reconstruct once disrupted. To address this problem, we developed See-Star, a hydrogel-based tissue clearing protocol to render the bodies of opaque and calcified invertebrates optically transparent while preserving their anatomy in an unperturbed state, facilitating molecular labeling and observation of intact organ systems. The resulting protocol can clear large (> 1 cm3) specimens to enable deep-tissue imaging, and is compatible with molecular techniques, such as immunohistochemistry and in situ hybridization to visualize protein and mRNA localization. To test the utility of this method, we performed a whole-mount imaging study of intact nervous systems in juvenile echinoderms and molluscs and demonstrate that See-Star allows for comparative studies to be extended far into development, facilitating insights into the anatomy of juveniles and adults that are usually not amenable to whole-mount imaging.
摘要：
许多无脊椎动物成年阶段的形态和发育模式的研究受到不透明结构的阻碍，比如贝壳，骨骼元素,和颜料颗粒，阻挡或折射光，需要切片观察内部特征。依赖于手术方法的研究中固有的挑战是切割组织是半破坏性的，精致的结构，例如神经网络中的轴突过程，一旦被破坏，重建在计算上都是有挑战性的。为了解决这个问题,我们开发了See-Star,基于水凝胶的组织清除方案，使不透明和钙化的无脊椎动物的身体光学透明，同时保持其解剖结构处于不受干扰的状态，促进完整器官系统的分子标记和观察。由此产生的协议可以清除大（>1cm3）标本，使深层组织成像，与分子技术兼容，例如免疫组织化学和原位杂交以可视化蛋白质和mRNA定位。要测试此方法的实用性，我们对幼年棘皮动物和软体动物的完整神经系统进行了全面的成像研究，并证明了See-Star允许将比较研究扩展到发育中，促进对青少年和成年人的解剖结构的见解，这些通常不适合全架成像。
