Beauty is more than skin deep: a non-invasive protocol for in vivo anatomical study using micro-CT

Microcomputed tomography (μCT) is a widely used tool in biomedical research, employed to investigate tissues and bone structures of small mammals in vivo. The application of in vivo μCT scanning in non-medical studies greatly lags behind the rapid advancements made in the biomedical field wherein the methodology has evolved to allow for longitudinal studies and eliminate the need to sacrifice the animal. Ecological and evolutionary studies often involve morphological measurements of a large sample of live animals; however, the potential of in vivo μCT imaging as a method for data acquisition has yet to be delineated.

Here, we describe a protocol for in vivo μCT imaging of the internal anatomy of reptiles and amphibians, commonly used study organisms in ecological and evolutionary research. We consider the skeletal and extraskeletal (i.e. osteoderms) bones of a lizard as a case study to elucidate the potential of in vivo μCT imaging. First, we explore the effects of various parameter settings on radiation dose, scan time and image quality. Secondly, we develop a protocol to immobilize and restrain study organisms during scanning without need for the administration of anaesthetics and compare the results of the in vivo protocol to images obtained post-mortem.

To immobilize animals, we replace the use of anaesthetics by cooling, thereby allowing the use of previously unsuitable rotating gantry μCT scanners that are readily available in scientific institutions. The resultant image quality of in vivo μCT scans is similar to that of post-mortem μCT scans, especially in the abdominal region. We discuss the effect of tube voltage, distance to X-ray source and metal filtration on radiation dose, and how these parameters could be altered to reduce the cumulative radiation dose while maintaining optimal image quality.

The proposed in vivo μCT protocol offers a new approach to acquire anatomical information for non-biomedical studies. We offer specific suggestions as to how the protocol can be employed to suit a variety of model organisms.

微型计算机断层扫描（Microcomputed tomography，μCT）是生物医学研究中广泛应用的工具，可用于活体小型哺乳动物的组织与骨骼结构研究。活体μCT扫描在非医学研究中的应用，远落后于生物医学领域的快速发展——后者已演进为可支持纵向研究且无需处死实验动物的方法学体系。生态学与进化研究通常需要对大量活体动物开展形态学测量，但活体μCT成像作为数据获取手段的潜力尚未得到充分阐明。

本文描述了一套针对爬行动物与两栖动物——生态学与进化研究中常用的模式生物——的内部解剖结构活体μCT成像方案。我们以蜥蜴的骨骼与骨骼外（如真皮骨）结构为案例研究，阐明活体μCT成像的应用潜力。首先，我们探究了多种参数设置对辐射剂量、扫描时长与图像质量的影响；其次，我们开发了一套无需使用麻醉剂即可在扫描过程中固定束缚实验动物的方案，并将活体成像方案的结果与死后扫描得到的图像进行了对比。

为固定实验动物，我们采用冷却手段替代麻醉剂，由此可使用此前不适用的旋转机架μCT扫描仪——这类设备在科研机构中易于获取。活体μCT扫描所得图像质量与死后μCT扫描结果相近，尤其在腹部区域。我们讨论了管电压、X射线源距离与金属滤过对辐射剂量的影响，以及如何在维持最优图像质量的前提下调整这些参数以降低累积辐射剂量。

本研究提出的活体μCT成像方案，为非生物医学研究领域的解剖信息获取提供了全新途径。我们还针对如何将该方案适配至多种模式生物给出了具体建议。