Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography

Silva, Juliana Martins de S. e; Zanette, Irène; Noël, Peter B.; Cardoso, Mateus Borba; Kimm, Melanie A.; Pfeiffer, Franz

doi:10.1038/srep14088

Cited by 82 publications

(56 citation statements)

References 29 publications

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“…Still, tissue discrimination (connective tissue or muscle fibers) is not easy at this resolution. Although both stains have been successfully used for micro‐CT studies (Metscher, ; Pauwels et al., ; Descamps et al., ; Silva et al., ), our results concur only partly with the literature. The distinct staining of the mostly collagenous body wall supports the assumed, but yet unproven binding preference of PTA for connective tissue (Metscher, ; Descamps et al., ).…”

Section: Discussionsupporting

confidence: 86%

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens)

2019

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Starfish (order: Asteroidea) possess a complex endoskeleton composed of thousands of calcareous ossicles. These ossicles are embedded in a body wall mostly consisting of a complex collagen fiber array. The combination of soft and hard tissue provides a challenge for detailed morphological and histological studies. As a consequence, very little is known about the general biomechanics of echinoderm endoskeletons and the possible role of ossicle shape in enabling or limiting skeletal movements. In this study, we used high-resolution X-ray microscopy to investigate individual ossicle shape in unprecedented detail. Our results show the variation of ossicle shape within ossicles of marginal, reticular and carinal type. Based on these results we propose an additional classification to categorize ossicles not only by shape but also by function into 'connecting' and 'node' ossicles. We also used soft tissue staining with phosphotungstic acid successfully and were able to visualize the ossicle ultrastructure at 2-lm resolution. We also identified two new joint types in the aboral skeleton (groove-on-groove joint) and between adambulacral ossicles (ball-and-socket joint). To demonstrate the possibilities of micro-computed tomographic methods in analyzing the biomechanics of echinoderm skeletons we exemplarily quantified changes in ossicle orientation for a bent ray for ambulacral ossicles. This study provides a first step for future biomechanical studies focusing on the interaction of ossicles and soft tissues during ray movements.

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Section: Discussionsupporting

confidence: 86%

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens)

2019

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“…Two types of dimensional quantification are possible: two dimensional in terms of perimeter, diameter and area, and three dimensional in terms of volume and surface (29). Moreover, as described in the consensus on bone histomorphometry (30) and extensively discussed in the accompanying BMAS nomenclature position paper, BMA parameters should be presented in relation to a reference region (31).…”

Section: Quantificationmentioning

confidence: 99%

“…Histological slicing and histomorphometry remain the gold standard for the ex vivo evaluation and characterization of biological tissues in general, and BMAT in particular, by measuring adipocyte cell size and cell number. However, histological assessment (sectioning, staining, imaging, and analysis) remains a challenging, time-consuming, and often costly technique (29). Moreover, spatial patterns as well as the spatial inter-relationship between different tissues within one sample (for example BMAT in relation to bone and vasculature) can be inaccurate or impossible.…”

Section: Ex Vivo Whole-bone Imagingmentioning

confidence: 99%

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society

et al. 2020

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The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced µCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt −/− , Kit W/W−v) and development of specific BMA deletion models would be highly desirable for this purpose.

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“…In spite of the high spatial resolution provided by X-ray micro-radiography and micro-CT, visualisation of soft tissue remains a challenging task due to the very low intrinsic attenuation contrast between the investigated structures. Consequently, for imaging of soft tissue with X-rays the application of a high-Z contrast agent is usually necessary 8 9 10 11 12 13 14 15 . To obtain a specific affinity of dedicated contrast agents to various tissue structures, a proper sample preparation protocol must be then followed.…”

Section: Virtual Histology By Means Of X-ray Imaging Techniquesmentioning

confidence: 99%

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector

Dudák

Žemlička

Karch

et al. 2016

Sci Rep

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Using dedicated contrast agents high-quality X-ray imaging of soft tissue structures with isotropic micrometre resolution has become feasible. This technique is frequently titled as virtual histology as it allows production of slices of tissue without destroying the sample. The use of contrast agents is, however, often an irreversible time-consuming procedure and despite the non-destructive principle of X-ray imaging, the sample is usually no longer usable for other research methods. In this work we present the application of recently developed large-area photon counting detector for high resolution X-ray micro-radiography and micro-tomography of whole ex-vivo ethanol-preserved mouse organs. The photon counting detectors provide dark-current-free quantum-counting operation enabling acquisition of data with virtually unlimited contrast-to-noise ratio (CNR). Thanks to the very high CNR even ethanol-only preserved soft-tissue samples without addition of any contrast agent can be visualized in great detail. As ethanol preservation is one of the standard steps of tissue fixation for histology, the presented method can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft-tissue visualisation.

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Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography

Cited by 82 publications

References 29 publications

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens)

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens)

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector

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