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Biological procedures online2020; 22; 13; doi: 10.1186/s12575-020-00126-4

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

Abstract: 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-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 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-α4 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.
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Publication Date: 2020-07-01 PubMed ID: 32624710PubMed Central: PMC7329428DOI: 10.1186/s12575-020-00126-4Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article presents a dual-method approach using fluorescence-based confocal microscopy and x-ray micro-computed tomography for 3D imaging of tendon tissues in both small and large animals, allowing for an improved understanding of tendon substructure composition and morphology.

Technology and Methodology

  • The study introduces a bimodal imaging procedure that combines fluorescence-based confocal microscopy and x-ray micro-computed tomography. This combination allows for a more detailed 3D visualization of adult tendons from any animal size, assisting in the analysis of their extracellular sub-structure and cellular composition.
  • Using immunolabeling and optical clearing, they detailed the distribution of laminin-α4—a cross-species marker of tendon basement membrane. The scans showcased this material throughout Achilles tendons in rats and superficial digital flexor tendon segments in equine specimens.
  • This process presented a detailed view of laminin-α4 within the tendon’s core, a complex network that mostly localizes within the interfascicular matrix compartment.
  • A specialized chemical drying process was applied to create contrast densities suitable for x-ray tomography, enabling the visualization and measurement of both fascicular and interfascicular matrix volume and thickness.
  • The study demonstrates that tissues previously labelled with fluorochrome can be reverse clarified prior to chemical drying, allowing both imaging modalities to be applied to a single sample.

Results and Conclusions

  • The method allowed researchers to discover an extensive network of laminin-α4 within the tendon structures. This level of complexity was unattainable via traditional 2D imaging.
  • The method of generating contrast for x-ray tomography through chemical drying was found to be simple, quick and distinctly superior to previously published methods.
  • Overall, the integration of the two imaging techniques provides a means to extract spatio-temporal data regarding tendons and facilitates the quantification of tendon substructures. By doing so, we can better understand the correlation between the morphology and function of tendons.

Cite This Article

APA
Marr N, Hopkinson M, Hibbert AP, Pitsillides AA, Thorpe CT. (2020). Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography. Biol Proced Online, 22, 13. https://doi.org/10.1186/s12575-020-00126-4

Publication

Biological procedures online
ISSN: 1480-9222
NlmUniqueID: 100963717
Country: England
Language: English
Volume: 22
Pages: 13
PII: 13

Researcher Affiliations

Marr, Neil
  • Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Hopkinson, Mark
  • Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Hibbert, Andrew P
  • Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Pitsillides, Andrew A
  • Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London, UK.
Thorpe, Chavaunne T
  • Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London, UK.

Grant Funding

  • 21216 / Versus Arthritis

Conflict of Interest Statement

Competing InterestsAll authors confirm they have no financial or non-financial competing interests.

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Citations

This article has been cited 10 times.
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