Callahan Sean, Crowe-Riddell Jenna M, Nagesan Ramon S, Gray Jaimi A, Davis Rabosky Alison R
Museum of Zoology University of Michigan Ann Arbor MI USA.
Department of Biology Eastern Michigan University Ypsilanti MI USA.
Ecol Evol. 2021 Jun 26;11(17):11587-11603. doi: 10.1002/ece3.7467. eCollection 2021 Sep.
Diffusible iodine-based contrast-enhanced computed tomography (diceCT) visualizes soft tissue from micro-CT (µCT) scans of specimens to uncover internal features and natural history information without incurring physical damage via dissection. Unlike hard-tissue imaging, taxonomic sampling within diceCT datasets is currently limited. To initiate best practices for diceCT in a nonmodel group, we outline a guide for staining and high-throughput µCT scanning in snakes. We scanned the entire body and one region of interest (i.e., head) for 23 specimens representing 23 species from the clades Aniliidae, Dipsadinae, Colubrinae, Elapidae, Lamprophiidae, and Viperidae. We generated 82 scans that include 1.25% Lugol's iodine stained (soft tissue) and unstained (skeletal) data for each specimen. We found that duration of optimal staining time increased linearly with body size; head radius was the best indicator. Postreconstruction of scans, optimal staining was evident by evenly distributed grayscale values and clear differentiation among soft-tissue anatomy. Under and over stained specimens produced poor contrast among soft tissues, which was often exacerbated by user bias during "digital dissections" (i.e., segmentation). Regardless, all scans produced usable data from which we assessed a range of downstream analytical applications within ecology and evolution (e.g., predator-prey interactions, life history, and morphological evolution). Ethanol destaining reversed the known effects of iodine on the exterior appearance of physical specimens, but required substantially more time than reported for other destaining methods. We discuss the feasibility of implementing diceCT techniques for a new user, including approximate financial and temporal commitments, required facilities, and potential effects of staining on specimens. We present the first high-throughput workflow for full-body skeletal and diceCT scanning in snakes, which can be generalized to any elongate vertebrates, and increases publicly available diceCT scans for reptiles by an order of magnitude.
基于可扩散碘的对比增强计算机断层扫描（diceCT）通过对标本进行微计算机断层扫描（µCT）来可视化软组织，从而揭示内部特征和自然历史信息，而无需通过解剖造成物理损伤。与硬组织成像不同，目前diceCT数据集中的分类采样受到限制。为了在非模式生物群体中开创diceCT的最佳实践，我们概述了一份蛇类染色和高通量µCT扫描指南。我们对代表Aniliidae、Dipsadinae、Colubrinae、Elapidae、Lamprophiidae和Viperidae等科23个物种的23个标本的全身和一个感兴趣区域（即头部）进行了扫描。我们生成了82次扫描，包括每个标本的1.25%卢戈氏碘染色（软组织）和未染色（骨骼）数据。我们发现最佳染色时间的持续时间与体型呈线性增加；头部半径是最佳指标。扫描重建后，通过均匀分布的灰度值和软组织解剖结构之间的清晰区分，可以明显看出最佳染色效果。染色不足和过度染色的标本在软组织之间产生的对比度较差，这在“数字解剖”（即分割）过程中常常因用户偏差而加剧。尽管如此，所有扫描都产生了可用数据，我们从中评估了生态和进化领域的一系列下游分析应用（例如，捕食者 - 猎物相互作用、生活史和形态进化）。乙醇脱色逆转了碘对物理标本外观的已知影响，但所需时间比其他脱色方法报道的要长得多。我们讨论了新用户实施diceCT技术的可行性，包括大致的财务和时间投入、所需设施以及染色对标本的潜在影响。我们展示了蛇类全身骨骼和diceCT扫描的首个高通量工作流程，该流程可推广到任何细长脊椎动物，并使公开可用的爬行动物diceCT扫描数量增加了一个数量级。
