MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues
Open accessAbstract
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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To image insects with conventional micro-CT, staining and/or drying is often required to discriminate between tissues.
“to image insects with conventional micro-CT, staining and/or drying is often required to discriminate between tissues 16 – 19”
From High-throughput phenomics of global ant biodiversity (2026) · cites this paper as [17]
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Cites
Hubbell, J. H., & Seltzer, S. M. (1995) · doi:10.6028/nist.ir.5632 · Cited by 2,033 · see citing works
Computed Tomography: Principles, Design, Artifacts, and Recent Advances
Hsieh, J. (2015) · doi:10.1117/3.2197756 · Cited by 1,412 · see citing works
Histological and Histochemical Methods: Theory and Practice
Kiernan, J. A. (1981) · Cited by 1,365 · see citing works
Principles and Techniques of Electron Microscopy: Biological Applications
Maugel, T. K., & Hayat, M. A. (1979) · Transactions of the American Microscopical Society · doi:10.2307/3226038 · Cited by 1,327 · see citing works
Histologic fixatives suitable for diagnostic light and electron microscopy.
McDowell, E. M., & Trump, B. F. (1976) · PubMed · Cited by 1,254 · see citing works
Histological and Histochemical Methods - Theory and practice
Malatesta, M. (2016) · European Journal of Histochemistry · doi:10.4081/ejh.2016.2639 · Cited by 1,182 · see citing works
Computed tomography : fundamentals, system technology, image quality, applications
Kalender, W. A. (2000) · Cited by 698 · see citing works
The structure and formula of 12-phosphotungstic acid
Keggin, J. F. (1934) · Proceedings of the Royal Society of London Series A Containing Papers of a Mathematical and Physical Character · doi:10.1098/rspa.1934.0035 · Cited by 627 · see citing works
Metscher, B. (2009) · Developmental Dynamics · doi:10.1002/dvdy.21857 · Cited by 609 · see citing works
Self-citationFormaldehyde, Formalin, Paraformaldehyde And Glutaraldehyde: What They Are And What They Do
Kiernan, J. A. (2000) · Microscopy Today · doi:10.1017/s1551929500057060 · Cited by 522 · see citing works
Humason's Animal tissue techniques
Presnell, J. K., Schreibman, M. P., & Humason, G. L. (1997) · Johns Hopkins University Press eBooks · Cited by 458 · see citing works
Synchrotron X-ray tomographic microscopy of fossil embryos
Donoghue, P. C. J., Bengtson, S., Dong, X.-P., Gostling, N. J., Huldtgren, T., Cunningham, J. A., Yin, C., Zhao, Y. J., Peng, F., & Stampanoni, M. (2006) · Nature · doi:10.1038/nature04890 · Cited by 305 · see citing works
Larabell, C. A., & Gros, M. A. L. (2003) · Molecular Biology of the Cell · doi:10.1091/mbc.e03-07-0522 · Cited by 283 · see citing works
Betz, O., Wegst, U. G. K., Weide, D., Heethoff, M., Helfen, L., LEE, W., & Cloetens, P. (2007) · Journal of Microscopy · doi:10.1111/j.1365-2818.2007.01785.x · Cited by 276 · see citing works
Principles and Techniques of Electron Microscopy: Biological Applications
Goode, M. D., & Hayat, M. A. (1973) · Transactions of the American Microscopical Society · doi:10.2307/3224936 · Cited by 254 · see citing works
X-ray tomography of whole cells
Gros, M. A. L., McDermott, G., & Larabell, C. A. (2005) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2005.08.008 · Cited by 226 · see citing works
Epigenetic interactions and the structure of phenotypic variation in the cranium
Hallgrímsson, B., Lieberman, D. E., Liu, W., Ford‐Hutchinson, A. F., & Jirik, F. R. (2007) · Evolution & Development · doi:10.1111/j.1525-142x.2006.00139.x · Cited by 200 · see citing works
Virtual Histology of Transgenic Mouse Embryos for High-Throughput Phenotyping
Johnson, J. T., Hansen, M. S., Wu, I. Q., Healy, L., Johnson, C. R., Jones, G., Capecchi, M. R., & Keller, C. (2006) · PLoS Genetics · doi:10.1371/journal.pgen.0020061 · Cited by 183 · see citing works
Weninger, W. J., Geyer, S. H., Mohun, T. J., Rasskin‐Gutman, D., Matsui, T., Ribeiro, I., Costa, L. D. F., Izpisúa‐Belmonte, J. C., & Müller, G. B. (2006) · Anatomy and Embryology · doi:10.1007/s00429-005-0073-x · Cited by 164 · see citing works
Sharpe, J. A. (2004) · Annual Review of Biomedical Engineering · doi:10.1146/annurev.bioeng.6.040803.140210 · Cited by 160 · see citing works
Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography
Parkinson, D. Y., McDermott, G., Etkin, L. D., et al. (2008) · Journal of Structural Biology · doi:10.1016/j.jsb.2008.02.003 · Cited by 158 · see citing works
Advances in Biological Structure, Function, and Physiology Using Synchrotron X-Ray Imaging
Westneat, M. W., Socha, J. J., & Lee, W.-K. (2008) · Annual Review of Physiology · doi:10.1146/annurev.physiol.70.113006.100434 · Cited by 147 · see citing works
Three‐dimensional computer analysis of white shark jaw mechanics: how hard can a great white bite?
Wroe, S. W., Huber, D. R., Lowry, M. B., et al. (2008) · Journal of Zoology · doi:10.1111/j.1469-7998.2008.00494.x · Cited by 135 · see citing works
BEUTEL, R. G., Ge, S., & Hörnschemeyer, T. (2007) · Cladistics · doi:10.1111/j.1096-0031.2007.00186.x · Cited by 126 · see citing works
High-throughput morphologic phenotyping of the mouse brain with magnetic resonance histology
Johnson, G. A., Ali-Sharief, A., Badea, A., Brandenburg, J., Cofer, G. P., Fubara, B. N., Gewalt, S. L., Hedlund, L. W., & Upchurch, L. (2007) · NeuroImage · doi:10.1016/j.neuroimage.2007.05.013 · Cited by 121 · see citing works
Head structures ofPriacma serrataleconte (coleptera, archostemata) inferred from X‐ray tomography
Hörnschemeyer, T., BEUTEL, R. G., & Pasop, F. (2002) · Journal of Morphology · doi:10.1002/jmor.1107 · Cited by 117 · see citing works
3D micro-CT imaging of the postmortem brain
de Crespigny, A. J., Bou-Reslan, H., Nishimura, M. C., PHILLIPS, H. S., Carano, R. A. D., & D'Arceuil, H. E. (2008) · Journal of Neuroscience Methods · doi:10.1016/j.jneumeth.2008.03.006 · Cited by 103 · see citing works
Imaging honey bee brain anatomy with micro-X-ray-computed tomography
Ribi, W. A., Senden, T. J., Sakellariou, A., Limaye, A. C., & Zhang, S. (2008) · Journal of Neuroscience Methods · doi:10.1016/j.jneumeth.2008.02.010 · Cited by 90 · see citing works
X-ray Microcomputer Tomography for the Study of Biomineralized Endo- and Exoskeletons of Animals
Neues, F., & Epple, M. (2008) · Chemical Reviews · doi:10.1021/cr078250m · Cited by 77 · see citing works
Micro-computer tomography and a renaissance of insect morphology
Friedrich, F., & BEUTEL, R. G. (2008) · Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE · doi:10.1117/12.794057 · Cited by 73 · see citing works
Parsons, T. E., Kristensen, E., Hornung, L., Diewert, V. M., Boyd, S. K., German, R. Z., & Hallgrímsson, B. (2008) · Journal of Anatomy · doi:10.1111/j.1469-7580.2007.00845.x · Cited by 73 · see citing works
Wirkner, C. S., & Prendini, L. (2007) · Journal of Morphology · doi:10.1002/jmor.10512 · Cited by 54 · see citing works
Golding, R. E., Ponder, W. F., & Byrne, M. (2008) · Journal of Morphology · doi:10.1002/jmor.10699 · Cited by 53 · see citing works
Digital Three-Dimensional Atlas of Quail Development Using High-Resolution MRI
Ruffins, S. W., Martin, M., Keough, L., et al. (2007) · The Scientific World JOURNAL · doi:10.1100/tsw.2007.125 · Cited by 53 · see citing works
Computed tomography imaging of the neuronal structure ofDrosophilabrain
Mizutani, R., Takeuchi, A., Hara, T., Uesugi, K., & Suzuki, Y. (2007) · Journal of Synchrotron Radiation · doi:10.1107/s0909049507009004 · Cited by 52 · see citing works
Alanentalo, T., Lorén, C. E., Larefalk, Å., et al. (2008) · Journal of Biomedical Optics · doi:10.1117/1.3000430 · Cited by 52 · see citing works
Wirkner, C. S., & Richter, S. (2004) · Microscopy Research and Technique · doi:10.1002/jemt.20076 · Cited by 47 · see citing works
Micro-CT as a novel technique for 3D reconstruction of molluscan anatomy
Golding, R. E., & Jones, A. S. (2007) · Molluscan Research · doi:10.11646/mr.27.3.2 · Cited by 41 · see citing works
Element-specific microtomographic imaging ofDrosophilabrain stained with high-Zprobes
Mizutani, R., Takeuchi, A., Akamatsu, G., Uesugi, K., & Suzuki, Y. (2008) · Journal of Synchrotron Radiation · doi:10.1107/s0909049508003725 · Cited by 21 · see citing works
Nagase, T., Sasazaki, Y., Kikuchi, T., et al. (2008) · Journal of Computer Assisted Tomography · doi:10.1097/rct.0b013e318157c5e2 · Cited by 17 · see citing works
Sharpe, J. A. (2008) · Springer eBooks · doi:10.1007/978-3-540-68993-5_9 · Cited by 13 · see citing works
Digital Three-Dimensional Atlas of Quail Development Using High-Resolution MRI
Ruffins, S. W., Martin, M. J., Keough, L., et al. (2007) · TSW Development & Embryology · doi:10.1100/tswde.2007.137 · Cited by 11 · see citing works
Quantitative 3-D Imaging of Eukaryotic Cells using Soft X-Ray Tomography
Parkinson, D., McDermott, G., Gros, M. L., et al. (2008) · Microscopy and Microanalysis · doi:10.1017/s1431927608084900 · Cited by 4 · see citing works
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PAUWELS, E., VAN LOO, D., CORNILLIE, P., BRABANT, L., & VAN HOOREBEKE, L. (2013) · Journal of Microscopy · doi:10.1111/jmi.12013 · Cited by 212 · see citing works
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Davies, T. G., Rahman, I. A., Lautenschlager, S., Cunningham, J. A., Asher, R. J., Barrett, P. M., Bates, K. T., Bengtson, S., Benson, R. B. J., Boyer, D. M., et al. (2017) · Proceedings of the Royal Society B: Biological Sciences · doi:10.1098/rspb.2017.0194 · Cited by 181 · see citing works
Self-citationSombke, A., Lipke, E., Michalik, P., Uhl, G., & Harzsch, S. (2015) · Journal of Comparative Neurology · doi:10.1002/cne.23741 · Cited by 144 · see citing works
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Koch, S., Tahara, R., Vasquez‐Correa, A., & Abouheif, E. (2021) · Journal of Experimental Zoology Part B: Molecular and Developmental Evolution · doi:10.1002/jez.b.23097 · Cited by 5 · see citing works
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- Authors
- Metscher, B. D.
- Affiliations
- University of Vienna (AT)
- Corresponding author
- Metscher, Brian D
- ORCID
- Metscher, Brian D
- Venue
- BMC Physiology
- Published
- 2009
- Volume
- 9
- Issue
- 1
- Pages
- 11
- ISSN
- 1472-6793 (ISSN-L)
- PMC
- PMC2717911
- OpenAlex
- W2161247859
- Cited by
- 1,086
- References
- 43
- Citations by year
- 2026: 51 · 2025: 52 · 2024: 81 · 2023: 74 · 2022: 75 · 2021: 92 · 2020: 91 · 2019: 101 · 2018: 96 · 2017: 88 · 2016: 62 · 2015: 49 · 2014: 58 · 2013: 44 · 2012: 29
- FWCI
- 22.90
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by
- Locations
- BMC Physiology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Phaidra (Universität Wien) (submittedVersion, cc-by-nc-nd)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- Universität Wien
- Topics
- Physical Sciences › Physics and Astronomy › Radiation › Advanced X-ray Imaging Techniques
- Health Sciences › Medicine › Urology › Comparative Animal Anatomy Studies
- Physical Sciences › Engineering › Biomedical Engineering › Advanced X-ray and CT Imaging
- Keywords
- X-ray microtomography, staining techniques, phosphotungstic acid, comparative morphology, tissue fixation, sample preparation, invertebrate morphology, muscle fibers, blood cells
- MeSH terms
- Animals, Contrast Media, Iodine, Minerals, Phosphotungstic Acid, Radiographic Image Enhancement, Staining and Labeling, Tomography, X-Ray Computed, Imaging, Three-Dimensional
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