Live imaging of whole invertebrates can be accomplished with X-ray micro-computed tomography (micro-CT) at 10-100 μm spatial resolution. However, image quality could be compromised by the movement of live subjects, producing image artefacts. We tested the feasibility of using CO2 gas to induce temporary full-immobilization of sufficient duration to image live insects based on their ability to tolerate hypoxic conditions. Additionally, we investigated the effects of these prolonged hypoxic conditions on several life history traits of a lepidopteran species. Live Colorado potato beetle (CPB) and true armyworm (TAW) adults were immobilized under a constant CO2 gas flow (0.5 L/min), and scanned using micro-CT (80 kVp; 450 μA). An L8 (24) orthogonal array (OA) was used to evaluate the effects of prolonged CO2-induced anesthesia on the recovery, longevity, and incidence of mating of TAW adults. The variable factors were age (immature and mature), sex (female and male), exposure time (3 and 7 h), and exposure regime (single and repeated). With this method, successful 3D reconstruction and visualizations of CPB and TAW adults were produced at 20 micron voxel spacing at an acceptable radiation dose and image noise level. From the inverse-square relationship found between the radiation doses and image noise levels, the optimal scanning protocol produced an entrance dose of 6.2 ± 0.04 Gy with images of 129.6 ± 5.1 HU noise level during a 2.7 h scan. Independent OA experiments indicated that CO2 gas did not result in death of exposed TAW adults, except when older males were exposed for longer durations. Exposure time and sex were more influential factors affecting recovery, longevity, and mating success than age and exposure regime following CO2 exposure. We have demonstrated that using CO2 gas during micro-CT imaging effectively induces safe, repeatable, whole-body, and temporary immobilization of live insects for 3D visualizations without motion artefacts. Moreover, we have shown that exposed TAW individuals made a full recovery with very little impact on subsequent longevity, and mating success post hypoxia. This method is applicable to other imaging modalities and could be used for routine exploratory and time-course studies, for repeated scanning of live and intact individuals.

中文翻译：

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使用二氧化碳气体引起的缺氧作为麻醉剂对活昆虫进行微CT成像，对某些随后的生活史特征的影响最小

整个无脊椎动物的实时成像可以通过X射线微计算机断层扫描（micro-CT）以10-100μm的空间分辨率完成。但是，活动对象的移动可能会损害图像质量，从而产生图像伪影。我们基于其耐受缺氧条件的能力，测试了使用CO2气体诱导足够长的时间进行暂时完全固定以使活昆虫成像的可行性。此外，我们调查了这些长期缺氧条件对鳞翅目物种的几种生活史特征的影响。将活的科罗拉多马铃薯甲虫（CPB）和真夜蛾（TAW）成虫固定在恒定的CO2气流下（0.5 L / min），并使用微型CT（80 kVp; 450μA）进行扫描。L8（24）正交阵列（OA）用于评估长时间的CO2诱导的麻醉对恢复的影响，TAW成虫的寿命和交配发生率。可变因素是年龄（未成熟和成熟），性别（男女），接触时间（3和7小时）和接触方式（单次和重复）。使用这种方法，可以在可接受的辐射剂量和图像噪声水平下以20微米体素间距成功完成CPB和TAW成人的3D重建和可视化。根据辐射剂量与图像噪声水平之间的平方反比关系，最佳扫描方案在2.7 h扫描过程中产生的入射剂量为6.2±0.04 Gy，图像噪声为129.6±5.1 HU。独立的OA实验表明，CO2气体不会导致暴露的TAW成人死亡，除非年龄较大的男性暴露时间更长。接触时间和性别是影响恢复，寿命，和接触成功的年龄和接触二氧化碳后的接触方式。我们已经证明，在微型CT成像过程中使用CO2气体可以有效地诱导昆虫的安全，可重复，全身和暂时固定，从而实现无运动伪影的3D可视化。此外，我们已经表明，暴露的TAW个体可以完全恢复，而对随后的寿命和缺氧后的交配成功几乎没有影响。该方法适用于其他成像方式，可用于常规探索性研究和时程研究，用于活体和完整个体的重复扫描。并临时固定活昆虫，以进行3D可视化而没有运动伪像。此外，我们已经表明，暴露的TAW个体可以完全恢复，而对随后的寿命和缺氧后的交配成功几乎没有影响。该方法适用于其他成像方式，可用于常规探索性研究和时程研究，用于活体和完整个体的重复扫描。并临时固定活昆虫，以进行3D可视化而没有运动伪像。此外，我们已经表明，暴露的TAW个体可以完全恢复，而对随后的寿命和缺氧后的交配成功几乎没有影响。该方法适用于其他成像方式，可用于常规探索性研究和时程研究，用于活体和完整个体的重复扫描。