Three-dimensional visualisation of the internal anatomy of the sparrowhawk (Accipiter nisus) forelimb using contrast-enhanced micro-computed tomography – ScienceOpen
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      Three-dimensional visualisation of the internal anatomy of the sparrowhawk ( Accipiter nisus) forelimb using contrast-enhanced micro-computed tomography

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      PeerJ
      PeerJ Inc.
      3D modelling, Wing, Muscle architecture, Staining, Birds

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          Abstract

          Background

          Gross dissection is a widespread method for studying animal anatomy, despite being highly destructive and time-consuming. X-ray computed tomography (CT) has been shown to be a non-destructive alternative for studying anatomical structures. However, in the past it has been limited to only being able to visualise mineralised tissues. In recent years, morphologists have started to use traditional X-ray contrast agents to allow the visualisation of soft tissue elements in the CT context. The aim of this project is to assess the ability of contrast-enhanced micro-CT (μCT) to construct a three-dimensional (3D) model of the musculoskeletal system of the bird wing and to quantify muscle geometry and any systematic changes due to shrinkage. We expect that this reconstruction can be used as an anatomical guide to the sparrowhawk wing musculature and form the basis of further biomechanical analysis of flight.

          Methods

          A 3% iodine-buffered formalin solution with a 25-day staining period was used to visualise the wing myology of the sparrowhawk ( Accipiter nisus). μCT scans of the wing were taken over the staining period until full penetration of the forelimb musculature by iodine was reached. A 3D model was reconstructed by manually segmenting out the individual elements of the avian wing using 3D visualisation software.

          Results

          Different patterns of contrast were observed over the duration of the staining treatment with the best results occurring after 25 days of staining. Staining made it possible to visualise and identify different elements of the soft tissue of the wing. Finally, a 3D reconstruction of the musculoskeletal system of the sparrowhawk wing is presented and numerical data of muscle geometry is compared to values obtained by dissection.

          Discussion

          Contrast-enhanced μCT allows the visualisation and identification of the wing myology of birds, including the smaller muscles in the hand, and provides a non-destructive way for quantifying muscle volume with an accuracy of 96.2%. By combining contrast-enhanced μCT with 3D visualisation techniques, it is possible to study the individual muscles of the forelimb in their original position and 3D design, which can be the basis of further biomechanical analysis. Because the stain can be washed out post analysis, this technique provides a means of obtaining quantitative muscle data from museum specimens non-destructively.

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          Most cited references36

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          Micro-computed tomography: Introducing new dimensions to taxonomy

          Abstract Continuous improvements in the resolution of three-dimensional imaging have led to an increased application of these techniques in conventional taxonomic research in recent years. Coupled with an ever increasing research effort in cybertaxonomy, three-dimensional imaging could give a boost to the development of virtual specimen collections, allowing rapid and simultaneous access to accurate virtual representations of type material. This paper explores the potential of micro-computed tomography (X-ray micro-tomography), a non-destructive three-dimensional imaging technique based on mapping X-ray attenuation in the scanned object, for supporting research in systematics and taxonomy. The subsequent use of these data as virtual type material, so-called “cybertypes”, and the creation of virtual collections lie at the core of this potential. Sample preparation, image acquisition, data processing and presentation of results are demonstrated using polychaetes (bristle worms), a representative taxon of macro-invertebrates, as a study object. Effects of the technique on the morphological, anatomical and molecular identity of the specimens are investigated. The paper evaluates the results and discusses the potential and the limitations of the technique for creating cybertypes. It also discusses the challenges that the community might face to establish virtual collections. Potential future applications of three-dimensional information in taxonomic research are outlined, including an outlook to new ways of producing, disseminating and publishing taxonomic information.
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            Concentration-dependent specimen shrinkage in iodine-enhanced microCT.

            Iodine potassium iodide (I2 KI) solution can be employed as a contrast agent for the visualisation of soft tissue structures in micro-computed tomography studies. This technique provides high resolution images of soft tissue non-destructively but initial studies suggest that the stain can cause substantial specimen shrinkage. The degree of specimen shrinkage, and potential deformation, is an important consideration when using the data for morphological studies. Here we quantify the macroscopic volume changes in mouse skeletal muscle, cardiac muscle and cerebellum as a result of immersion in the common fixatives 10% phosphate-buffered formal saline, 70% ethanol and 3% glutaraldehyde, compared with I2 KI staining solution at concentrations of 2, 6, 10 and 20%. Immersion in the I2 KI solution resulted in dramatic changes of tissue volume, which were far larger than the shrinkage from formalin fixation alone. The degree of macroscopic change was most dependent upon the I2 KI concentration, with severe shrinkage of 70% seen in solutions of 20% I2 KI after 14 days' incubation. When using this technique care needs to be taken to use the lowest concentration that will give adequate contrast to minimise artefacts due to shrinkage. © 2013 Anatomical Society.
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              The songbird syrinx morphome: a three-dimensional, high-resolution, interactive morphological map of the zebra finch vocal organ

              Background Like human infants, songbirds learn their species-specific vocalizations through imitation learning. The birdsong system has emerged as a widely used experimental animal model for understanding the underlying neural mechanisms responsible for vocal production learning. However, how neural impulses are translated into the precise motor behavior of the complex vocal organ (syrinx) to create song is poorly understood. First and foremost, we lack a detailed understanding of syringeal morphology. Results To fill this gap we combined non-invasive (high-field magnetic resonance imaging and micro-computed tomography) and invasive techniques (histology and micro-dissection) to construct the annotated high-resolution three-dimensional dataset, or morphome, of the zebra finch (Taeniopygia guttata) syrinx. We identified and annotated syringeal cartilage, bone and musculature in situ in unprecedented detail. We provide interactive three-dimensional models that greatly improve the communication of complex morphological data and our understanding of syringeal function in general. Conclusions Our results show that the syringeal skeleton is optimized for low weight driven by physiological constraints on song production. The present refinement of muscle organization and identity elucidates how apposed muscles actuate different syringeal elements. Our dataset allows for more precise predictions about muscle co-activation and synergies and has important implications for muscle activity and stimulation experiments. We also demonstrate how the syrinx can be stabilized during song to reduce mechanical noise and, as such, enhance repetitive execution of stereotypic motor patterns. In addition, we identify a cartilaginous structure suited to play a crucial role in the uncoupling of sound frequency and amplitude control, which permits a novel explanation of the evolutionary success of songbirds.
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                Author and article information

                Contributors
                Journal
                PeerJ
                PeerJ
                PeerJ
                PeerJ
                PeerJ
                PeerJ Inc. (San Francisco, USA )
                2167-8359
                15 March 2017
                2017
                : 5
                : e3039
                Affiliations
                Faculty of Science and Engineering, University of Manchester , Manchester, UK
                Author information
                http://orcid.org/0000-0002-2913-5406
                Article
                3039
                10.7717/peerj.3039
                5356476
                28316884
                be7cde36-51d1-4fe6-8ff1-c38f2ec8d697
                © 2017 Bribiesca-Contreras and Sellers

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

                History
                : 5 December 2016
                : 27 January 2017
                Funding
                Funded by: Consejo Nacional de Ciencia y Tecnologia (CONACyT)
                This work was supported by a scholarship provided to Fernanda Bribiesca-Contreras by the Consejo Nacional de Ciencia y Tecnologia (CONACyT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Computational Biology
                Zoology
                Radiology and Medical Imaging

                3d modelling,wing,muscle architecture,staining,birds
                3d modelling, wing, muscle architecture, staining, birds

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