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      MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues

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      BMC Physiology

      BioMed Central

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          Abstract

          Background

          Comparative, functional, and developmental studies of animal morphology require accurate visualization of three-dimensional structures, but few widely applicable methods exist for non-destructive whole-volume imaging of animal tissues. Quantitative studies in particular require accurately aligned and calibrated volume images of animal structures. X-ray microtomography (microCT) has the potential to produce quantitative 3D images of small biological samples, but its widespread use for non-mineralized tissues has been limited by the low x-ray contrast of soft tissues. Although osmium staining and a few other techniques have been used for contrast enhancement, generally useful methods for microCT imaging for comparative morphology are still lacking.

          Results

          Several very simple and versatile staining methods are presented for microCT imaging of animal soft tissues, along with advice on tissue fixation and sample preparation. The stains, based on inorganic iodine and phosphotungstic acid, are easier to handle and much less toxic than osmium, and they produce high-contrast x-ray images of a wide variety of soft tissues. The breadth of possible applications is illustrated with a few microCT images of model and non-model animals, including volume and section images of vertebrates, embryos, insects, and other invertebrates. Each image dataset contains x-ray absorbance values for every point in the imaged volume, and objects as small as individual muscle fibers and single blood cells can be resolved in their original locations and orientations within the sample.

          Conclusion

          With very simple contrast staining, microCT imaging can produce quantitative, high-resolution, high-contrast volume images of animal soft tissues, without destroying the specimens and with possibilities of combining with other preparation and imaging methods. Such images are expected to be useful in comparative, developmental, functional, and quantitative studies of morphology.

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          Most cited references 42

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          MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions.

           B D Metscher (2009)
          Understanding developmental processes requires accurate visualization and parameterization of three-dimensional embryos. Tomographic imaging methods offer automatically aligned and calibrated volumetric images, but the usefulness of X-ray CT imaging for developmental biology has been limited by the low inherent contrast of embryonic tissues. Here, I demonstrate simple staining methods that allow high-contrast imaging of embryonic tissues at histological resolutions using a commercial microCT system. Quantitative comparisons of images of chick embryos treated with different contrast agents show that three very simple methods using inorganic iodine and phosphotungstic acid produce overall contrast and differential tissue contrast for X-ray imaging at least as high as that obtained with osmium. The stains can be used after any common fixation and after storage in aqueous or alcoholic media, and on a wide variety of species. These methods establish microCT imaging as a useful tool for comparative developmental studies, embryo phenotyping, and quantitative modeling of development. (c) 2009 Wiley-Liss, Inc.
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            Synchrotron X-ray tomographic microscopy of fossil embryos.

             Fan Peng,  Xi Dong,  Z Yue (2006)
            Fossilized embryos from the late Neoproterozoic and earliest Phanerozoic have caused much excitement because they preserve the earliest stages of embryology of animals that represent the initial diversification of metazoans. However, the potential of this material has not been fully realized because of reliance on traditional, non-destructive methods that allow analysis of exposed surfaces only, and destructive methods that preserve only a single two-dimensional view of the interior of the specimen. Here, we have applied synchrotron-radiation X-ray tomographic microscopy (SRXTM), obtaining complete three-dimensional recordings at submicrometre resolution. The embryos are preserved by early diagenetic impregnation and encrustation with calcium phosphate, and differences in X-ray attenuation provide information about the distribution of these two diagenetic phases. Three-dimensional visualization of blastomere arrangement and diagenetic cement in cleavage embryos resolves outstanding questions about their nature, including the identity of the columnar blastomeres. The anterior and posterior anatomy of embryos of the bilaterian worm-like Markuelia confirms its position as a scalidophoran, providing new insights into body-plan assembly among constituent phyla. The structure of the developing germ band in another bilaterian, Pseudooides, indicates a unique mode of germ-band development. SRXTM provides a method of non-invasive analysis that rivals the resolution achieved even by destructive methods, probing the very limits of fossilization and providing insight into embryology during the emergence of metazoan phyla.
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              Histological and Histochemical Methods: Theory and Practice

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                Author and article information

                Affiliations
                [1 ]Department of Theoretical Biology, Gerd Müller, head, University of Vienna, Althanstraße 14, 1090 Austria
                Contributors
                Journal
                BMC Physiol
                BMC Physiology
                BioMed Central
                1472-6793
                2009
                22 June 2009
                : 9
                : 11
                2717911
                1472-6793-9-11
                19545439
                10.1186/1472-6793-9-11
                Copyright © 2009 Metscher; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Methodology Article

                Anatomy & Physiology

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