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提升了我们可视化、研究并数字化共享复杂且往往细微的解剖关联的能力。近年来，弥散碘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，开始为我们解析后生动物（Metazoa）各类软组织复合体的精细结构提供了此前无法企及的认知维度。作为造影剂，溶解态碘可深入渗透至经过固定的标本中，与后生动物软组织中天然存在的脂肪与碳水化合物结合，以可预测的方式提升这些组织的放射密度。如同当前的“金标准”磁共振成像（magnetic resonance imaging），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。以大脑为例，diceCT可将有髓神经纤维束与无髓鞘的大脑皮层、神经核团及神经节区分开来，并能在前所未有的空间尺度上实现这些结构解剖关联的三维可视化。本研究中，我们展示了diceCT在快速可视化脊椎动物（vertebrates）大脑内外解剖结构方面的应用价值——同时还能完整呈现颅骨骨骼、外周神经完整且未受干扰的通路，直至其所支配的靶器官。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谱进化研究而对大量标本进行成像的最佳操作规范进行了阐述。