Dataset Information

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.

ABSTRACT: Background:Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. Results:Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-?4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5?mm segments. This revealed a complex network of laminin-?4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. Conclusions:Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-?4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

SUBMITTER: Marr N

PROVIDER: S-EPMC7329428 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.

Marr Neil N Hopkinson Mark M Hibbert Andrew P AP Pitsillides Andrew A AA Thorpe Chavaunne T CT

Biological procedures online 20200701

<h4>Background</h4>Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species.<h4>Results</h4>Using fluorescent immunola ...[more]

PMID: 32624710

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Project description:BackgroundSexual selection has been considered to promote diversification and speciation. Sexually dimorphic species have been used to explore the supposed effect, however, with mixed results. In dwarf spiders (Erigoninae), many species are sexually dimorphic-males possess marked prosomal modifications. These male traits vary from moderate elevations to bizarre shapes in various prosomal regions. Previous studies established that male dwarf spiders produce substances in these prosomal modifications that are taken up by the females. These substances can act as nuptial gifts, which increase the mating probability of males and the oviposition rate in females. Therefore, these dimorphic traits have evolved in the context of sexual selection. Here, we explore the evolutionary lability of this gustatory trait complex with the aim of assessing the role of this trait complex in species divergence by investigating (1) if erigonine modified prosomata are inherently linked to nuptial-gift-producing glands, (2) if the evolution of the glands evolution preceded that of the modified prosomal shapes, and by assessing (3) the occurrence of convergent/divergent evolution and cryptic differentiation.ResultsWe reconstructed the position and extent of the glandular tissue along with the muscular anatomy in the anterior part of the prosoma of 76 erigonine spiders and three outgroup species using X-ray micro-computed tomography. In all but one case, modified prosomata are associated with gustatory glands. We incorporated the location of glands and muscles into an existing matrix of somatic and genitalic morphological traits of these taxa and reanalyzed their phylogenetic relationship. Our analysis supports that the possession of glandular equipment is the ancestral state and that the manifold modifications of the prosomal shape have evolved convergently multiple times. We found differences in gland position between species with both modified and unmodified prosomata, and reported on seven cases of gland loss.ConclusionsOur findings suggest that the occurrence of gustatory glands in sexually monomorphic ancestors has set the stage for the evolution of diverse dimorphic external modifications in dwarf spiders. Differences among congeners suggest that the gland position is highly susceptible to evolutionary changes. The multiple incidences might reflect costs of glandular tissue maintenance and nuptial feeding. Our results indicate divergent evolutionary patterns of gustatory-courtship-related traits, and thus a likely facilitating effect of sexual selection on speciation.

Dataset Information

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.

Publications

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.

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