Supplementary Material for: The Utility of DiceCT Imaging for High-Throughput Comparative Neuroanatomical Studies

Advancements in imaging techniques have drastically improved our ability to visualize, study, and digitally share complex, often minute, anatomical relationships. The recent adoption of soft-tissue X-ray imaging techniques, such as diffusible iodine-based contrast-enhanced computed tomography (diceCT), is beginning to offer previously unattainable insights into the detailed configurations of soft- tissue complexes across Metazoa. As a contrast agent, dissolved iodine diffuses deeply throughout preserved specimens to bind fats and carbohydrates that are natural ly present within metazoan soft tissues, increasing the radiodensities of these tissues in predictable ways. Like the current “gold standard” of magnetic resonance imaging, diceCT does not require physical dissection and can differentiate between the lipid content of myelinated versus nonmyelinated tissues, thereby offering great potential for neuroanatomical studies. Within the brain, for example, diceCT distinguishes myelinated fiber tracts from unmyelinated cortices, nuclei, and ganglia and allows three-dimensional visualization of their anatomical interrelationships at previously unrealized spatial scales. In this study, we illustrate the utility of diceCT for the rapid visualization of both external and internal brain anatomy in vertebrates – alongside the intact bones of the skull and the complete, undisturbed pathways of peripheral nerves, up to and including the target organs that they innervate. We demonstrate the transformative potential of this technique for developing high-resolution neuroanatomical datasets and describe best practices for imaging large numbers of specimens for broad evolutionary studies across vertebrates.

成像技术的进步极大地提升了我们可视化、研究以及数字化共享复杂且往往细微的解剖结构关系的能力。近年来，弥散碘对比增强计算机断层扫描（diffusible iodine-based contrast-enhanced computed tomography, diceCT）这类软组织X射线成像技术的应用，开始为解析后生动物（Metazoa）各类软组织复合体的精细构型提供了此前难以企及的视角。作为对比剂，溶解态碘可深入扩散至已固定的标本中，与后生动物软组织内天然存在的脂肪和碳水化合物结合，以可预测的方式提升这些组织的放射密度。与当前磁共振成像的“金标准”相比，diceCT无需进行实体解剖，还可区分有髓鞘与无髓鞘组织的脂质含量，因此在神经解剖学研究中具备巨大应用潜力。例如在大脑中，diceCT可将有髓鞘纤维束与无髓鞘皮层、神经核及神经节区分开来，并能在前所未有的空间尺度上实现二者解剖关联的三维可视化。本研究展示了diceCT在快速可视化脊椎动物大脑外部与内部解剖结构方面的应用价值——同时可完整呈现颅骨骨骼以及外周神经完整且未受干扰的通路，直至其支配的靶器官。我们证实了该技术在构建高分辨率神经解剖学数据集方面的变革性潜力，并针对为开展跨脊椎动物的广泛演化研究而成像大量标本的最佳实践流程进行了阐述。