Department of Zoology, Institute of Biology, University of Kassel, Kassel, Germany.
Departamento de Zoologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
J Microsc. 2018 Jun;270(3):343-358. doi: 10.1111/jmi.12688. Epub 2018 Feb 22.
Non-invasive imaging techniques like X-ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external morphology of organisms. Nevertheless, the effect of different staining approaches required for this method on samples lacking mineralized tissues, such as soft-bodied invertebrates, remains understudied. Herein, we used synchrotron radiation-based X-ray micro-computed tomography to compare the effects of commonly used contrasting approaches on onychophorans - soft-bodied invertebrates important for studying animal evolution. Representatives of Euperipatoides rowelli were stained with osmium tetroxide (vapour or solution), ruthenium red, phosphotungstic acid, or iodine. Unstained specimens were imaged using both standard attenuation-based and differential phase-contrast setups to simulate analyses with museum material. Our comparative qualitative analyses of several tissue types demonstrate that osmium tetroxide provides the best overall tissue contrast in onychophorans, whereas the remaining staining agents rather favour the visualisation of specific tissues and/or structures. Quantitative analyses using signal-to-noise ratio measurements show that the level of image noise may vary according to the staining agent and scanning medium selected. Furthermore, box-and-whisker plots revealed substantial overlap in grey values among structures in all datasets, suggesting that a combination of semiautomatic and manual segmentation of structures is required for comprehensive 3D reconstructions of Onychophora, irrespective of the approach selected. Our results show that X-ray micro-computed tomography is a promising technique for studying onychophorans and, despite the benefits and disadvantages of different staining agents for specific tissues/structures, this method retrieves informative data that may eventually help address evolutionary questions long associated with Onychophora.
非侵入性成像技术，如 X 射线计算机断层扫描，在动物学中已经变得非常流行，因为它们可以同时对生物体的内部和外部形态进行成像。然而，对于缺乏矿化组织的样本，如软体无脊椎动物，这种方法所需的不同染色方法的效果仍然研究不足。在这里，我们使用基于同步辐射的 X 射线微计算机断层扫描来比较常用对比方法对软体无脊椎动物 - 对研究动物进化很重要的有爪动物的影响。Euperipatoides rowelli 的代表用锇四氧化物（蒸气或溶液）、钌红、磷钨酸或碘进行染色。未染色的标本使用标准衰减基础和差分相衬设置进行成像，以模拟对博物馆材料的分析。我们对几种组织类型的比较定性分析表明，锇四氧化物在有爪动物中提供了最佳的整体组织对比度，而其余的染色剂则更有利于特定组织和/或结构的可视化。使用信噪比测量的定量分析表明，图像噪声水平可能根据所选的染色剂和扫描介质而变化。此外，箱线图显示所有数据集结构的灰度值之间存在很大的重叠，这表明无论选择哪种方法，都需要半自动和手动分割结构的组合，才能对有爪动物进行全面的 3D 重建。我们的结果表明，X 射线微计算机断层扫描是研究有爪动物的一种很有前途的技术，尽管不同的染色剂对特定组织/结构有优缺点，但这种方法可以获取有信息的数据，最终可能有助于解决长期以来与有爪动物相关的进化问题。
