Characterisation of cuticular inflation development and ultrastructure in Trichuris muris using correlative X-ray computed tomography and electron microscopy

O'Sullivan, James D B; Cruickshank, Sheena M.; Starborg, Tobias; Withers, Philip J.; Else, Kathryn J.

doi:10.1038/s41598-020-61916-0

Cited by 15 publications

(7 citation statements)

References 31 publications

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“…In order to produce effective imaging results, osmium tetroxide staining proved particularly suitable in contrasting adult worms against the host epithelium. We speculate that this is due to the membrane-rich and therefore highly osmophilic nature of the stichosome ultrastructure, which has been noted previously by our lab via 3D electron microscopy [31]. Given the growing interest in using microCT for imaging biological soft tissues [32], and the need for the development of effective soft tissue imaging protocols, we have demonstrated OsO4 as a useful and specific stain for Trichuris, which may even be extended to the CT visualization of other highly membranous cellular structures such as acinar and beta cells of the pancreas and plasma cells [33][34][35].…”

Section: Worms Exhibit "Head Down" Behaviorsupporting

confidence: 68%

Morphological variability in the mucosal attachment site ofTrichuris murisrevealed by X-ray microcomputed tomography

O'Sullivan

Cruickshank

Withers

et al. 2021

Preprint

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Parasitic infections can be challenging to study, because light and electron microscopy are often limited in visualising complex and inaccessible attachment sites. Exemplifying this, Trichuris spp. inhabits a tunnel of epithelial cells within the host caecum and colon. A significant global burden of this infection persists partly because available anthelminthics lack efficacy, although the mechanisms underlying this remain unknown. Consequently, there is a need to pioneer new approaches to better characterize the parasite niche within the host and investigate how variation in its morphology and integrity may contribute to resistance to therapeutic intervention. To address these aims, we exploited 3D X-ray micro-computed tomography (microCT) to image the mouse whipworm T. muris in caeca of wild-type C57BL/6 and SCID mice ex vivo. Using osmium tetroxide staining to effectively enhance contrast of worms, we found that a subset exhibited preferential positioning towards the bases of the intestinal crypts. Moreover, in one rare event, we demonstrate whipworm traversal of the lamina propria. This morphological variability contradicts widely accepted conclusions from conventional microscopy of the parasite niche, showing Trichuris in close contact with the host proliferative and immune compartments that may facilitate immunomodulation. Furthermore, by using a skeletonization-based approach we demonstrate considerable variation in tunnel length and integrity which may represent an indicator of tunnel “health”. The qualitative and quantitative observations provide a new morphological point of reference for future in vitro study of Trichuris-host interactions and highlight the potential of microCT to more accurately characterise enigmatic host-parasite interactions.Author SummaryParasites are often difficult to observe once established within host tissues, presenting a barrier to biological understanding and therapeutic innovation. Whipworms (Trichuris spp.) affect 500 million people worldwide, causing significant disability, and appear partially resistant to widely used “deworming” drugs. However, the inaccessibility of worms within the cells of the host intestine makes them highly challenging to image and study. By investigating Trichuris attachment sites in 3D, using X-ray micro-computed tomography, we found that the niche is highly variable in size and, contrary to reports in all previous studies, can also penetrate different layers of intestinal tissue. By showing that worms are positioned much closer to host immune cells that previously appreciated, we provide a morphological reference point for future studies on how Trichuris effectively avoids clearance by the host. The non-invasive imaging approach used represents an excellent opportunity to clarify the lifecycles of other difficult-to-study parasites.

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Section: Worms Exhibit "Head Down" Behaviorsupporting

confidence: 68%

Morphological variability in the mucosal attachment site ofTrichuris murisrevealed by X-ray microcomputed tomography

O'Sullivan

Cruickshank

Withers

et al. 2021

Preprint

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“…This approach has been demonstrated with FIB-SEM nanotomography of materials (117), in bridging brightfield light microscopy to TEM in the assaying of adipose tissue (118), as well as through the use of geneticallyencoded tags in murine brain sections, which were subsequently imaged via serial block face EM (25). More recent work has utilized XRM-dependent correlative approaches to an increasingly wide range of targets, from exploring ultrastructural properties of parasitic worms in the small bowel (119), to correlating histological and TEM imaging of Araneae papal organs (120), and to link intravital microscopy studies of mouse cerebral vasculature to FIB-SEM nanotomographic analysis (121). An added advantage of the high-resolution, mesoscale imaging, and speed of XRM acquisition is the ability to employ the technique as a region of interest localization tool.…”

Section: Correlative Approachesmentioning

confidence: 99%

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Bayguinov

Fisher

2020

Journal of Biological Chemistry

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Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes have been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, whilst insightful, are nonetheless limited by either restrictions in resolution or by the sheer complexity of generating multi-dimensional data. Recent advances in the use and application of X-rays to acquire micro- and nano-tomographic datasets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the sub-cellular analyses of unstained vitrified cells, three-dimensional localization of specific protein targets, and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles and applications of both soft X-ray tomography and X-ray microscopy, and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches, and detail how they have become integral in correlative microscopy workflows.

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“…morphology, chemistry, structure) are linked. This approach is currently a developing application in human-made materials research [30][31][32][33] and life/biological science [34][35][36][37] , and despite a few geological [38][39][40] and more recently specific soil science examples 41,42 , the method has not been applied to soil weathering.…”

Section: Introductionmentioning

confidence: 99%

Correlative Microscopy: a tool for understanding soil weathering in modern analogues of early terrestrial biospheres

Mitchell¹,

Davies²,

Kenrick³

et al. 2021

Preprint

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Correlative imaging provides a method of investigating complex systems by combining analytical (chemistry) and imaging (tomography) information across dimensions (2D-3D) and scales (centimetres-nanometres). We studied weathering processes in a modern cryptogamic ground cover (CGC) from Iceland, containing early colonizing, and evolutionary ancient, communities of mosses, lichens, fungi, and bacteria. Targeted multi-scale X-ray Microscopy (XRM) of a grain in-situ within a soil core revealed networks of surficial and internal features (tunnels) originating from organic-rich surface holes. Further targeted 2D grain characterisation by optical microscopy (OM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (SEM-EDS), following an intermediate manual correlative preparation step, revealed Fe-rich nodules within the tunnels. Finally, nanotomographic imaging by focussed ion beam microscopy (FIB-SEM) revealed coccoid and filamentous-like structures within subsurface tunnels, as well as accumulations of Fe and S in grain surface crusts, which may represent a biological rock varnish/glaze. We attribute these features to biological processes. This work highlights the advantages and novelty of the correlative imaging approach, across scales, dimensions, and modes, to investigate biological weathering processes. Further, we demonstrate correlative microscopy as a means of identifying fingerprints of biological communities, which could be used in the geologic rock record and on extra-terrestrial bodies.

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Characterisation of cuticular inflation development and ultrastructure in Trichuris muris using correlative X-ray computed tomography and electron microscopy

Cited by 15 publications

References 31 publications

Morphological variability in the mucosal attachment site ofTrichuris murisrevealed by X-ray microcomputed tomography

Morphological variability in the mucosal attachment site ofTrichuris murisrevealed by X-ray microcomputed tomography

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Correlative Microscopy: a tool for understanding soil weathering in modern analogues of early terrestrial biospheres

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