Evaluation of contrasting techniques for X‐ray imaging of velvet worms (Onychophora)

Abstract

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.

Nucleus-specific X-ray stain for 3D virtual histology

“…The hematein-based staining protocol allows for high-resolution CT visualization specifically for cell nuclei within soft tissue down to the sub-micron range, which has not been possible so far applying other common staining methods used in microCT technology 11–16 . Nondestructive generation of virtual histological slices that are comparable in contrast and resolution to conventional histological data is rendered possible in combination with the recently developed nanoCT devices 6–10,26–28 .…”

“…Developments on the technological side allow for comparative resolution to 2D conventional histology with devices ranging from large particle accelerators 3–5 to laboratory X-ray devices 6–10 . Next to the technological requirements needed for attenuation-based X-ray microscopy, X-ray suitable staining agents such as phosphotungstic acid (PTA), iodine potassium iodide (IKI) or iodine in ethanol (I2E)/iodine in methanol (I2M) are an important aspect 11–16 . Nonetheless, the availability of staining agents that (i) target a specific biological morphology, (ii) stain homogenously and completely, (iii) are easy to handle, (iv) are speedily penetrating the tissue without creating artefacts such as diffusion rings, (v) are suitable for large and dense tissue samples, and (vi) are fully compatible with histology, is currently very limited 17 .…”

Nanoscopic X-ray tomography for correlative microscopy of a small meiofaunal sea-cucumber

“…In this case, a centre shift correction was sufficient to achieve the best possible image quality. The osmium tetroxide post-fixation binds to lipids and proteins and thereby acts as an overview stain in X-ray imaging 4,10,21 . It generated an adequate overall contrast to access the 3D morphology of the sea cucumber.…”

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

“…(2016) and also this study—it might be considered when the sample tissue is thin or removal of parts of the exoskeleton is possible (to facilitate stain perfusion). While osmium tetroxide (OsO 4 ) has been a popular staining agent in previous X‐ray CT studies (Jahn et al., 2018; Kim et al., 2012; Metscher, 2009a,b; Ribi, Senden, Sakellariou, Limaye, & Zhang, 2008), we did not consider it because of its undesirable toxicity, high costs, limited tissue penetration, and failure to work properly in tissues preserved in alcohol (Hayat, 1970; Metscher, 2009a; Smith, Carson, & Ferguson, 1974; Smith et al., 2016). …”