Soft tissue discrimination with contrast agents using micro-CT scanning

Emilie Descamps, Alicja Sochacka, Barbara De Kegel, Denis Van Loo, Luc Van Hoorebeke, Dominique Adriaens


The use of high resolution, three-dimensional visualization has been receiving growing interest within life sciences, with non-invasive imaging tools becoming more readily accessible. Although initially useful for visualizing mineralized tissues, recent developments are promising for studying soft tissues as well. Especially for micro-CT scanning, several X-ray contrast enhancers are performant in sufficiently contrasting soft tissue organ systems by a different attenuation strength of X-rays. Overall visualization of soft tissue organs has proven to be possible, although the tissue-specific capacities of these enhancers remain unclear. In this study, we tested several contrast agents for their usefulness to discriminate between tissue types and organs, using three model organisms (mouse, zebrafish and Xenopus). Specimens were stained with osmium tetroxide (OsO4), phosphomolybdic acid (PMA) and phosphotungstic acid (PTA), and were scanned using high resolution microtomography. The contrasting potentials between tissue types and organs are described based on volume renderings and virtual sections. In general, PTA and PMA appeared to allow better discrimination. Especially epithelial structures, cell-dense brain regions, liver, lung and blood could be easily distinguished. The PMA yielded the best results, allowing discrimination even at the level of cell layers. Our results show that those staining techniques combined with micro-CT imaging have good potential for use in future research in life sciences.


3D visualization; micro-CT scanning; soft tissue; contrast agents; vertebrates

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Allen Institute for Brain Science. (2014). “Allen mouse brain atlas.” from

Aoyagi H, Iwasaki SI & Nakamura K (2013). Three-dimensional observation of mouse tongue muscles using micro-computed tomography. Odontology.

Aoyagi H, Tsuchikawa K & Iwasaki S (2010). Three-dimensional observation of the mouse embryo by micro-computed tomography: Composition of the trigeminal ganglion. Odontology 98(1): 26-30.

Baron RL (1994). Understanding and optimizing use of contrast material for ct of the liver. American Journal of Roentgenology 163(2): 323-331.

Batenburg JJ & Haagsman HP (1998). The lipids of pulmonary surfactant: Dynamics and interactions with proteins. Progress in Lipid Research 37(4): 235-276.

Baverstock H, Jeffery NS & Cobb SN (2013). The morphology of the mouse masticatory musculature. Journal of Anatomy 223(1): 46-60.

Betz O, Wegst U, Weide D, Heethoff M, Helfen L, Lee WAHK & Cloetens P (2007). Imaging applications of synchrotron x-ray phase-contrast microtomography in biological morphology and biomaterials science. I. General aspects of the technique and its advantages in the analysis of millimetre sized arthropod structure. Journal of Microscopy 227(1): 51-71.

Bock WJ, Shear, R.C. (1972). A staining method for gross dissection of vertebrate muscles. Anat.Anz.Bd. 130: 222-227.

Boistel R, Swoger J, Kržič U, Fernandez V, Gillet B & Reynaud EG (2011). The future of three-dimensional microscopic imaging in marine biology. Marine Ecology 32(4): 438-452.

Brunelli E, Perrotta E & Tripepi S (2004). Ultrastructure and development of the gills in rana dalmatina (amphibia, anura). Zoomorphology 123(4): 203-211.

Burkl W & Schiechl H (1968). A study of osmium tetroxide fixation. Journal of Histochemistry & Cytochemistry 16(3): 157-161.

Buytaert J, Descamps E, Adriaens D & Dirckx JJJ (2012). The opfos microscopy family: High-resolution optical-sectioning of biomedical specimens. Anatomy Research International 2012: 9 pages.

Carden A, Rajachar RM, Morris MD & Kohn DH (2003). Ultrastructural changes accompanying the mechanical deformation of bone tissue: A raman imaging study. Calcif. Tissue Int. 72: 166-175.

Carson FL & Hladik C (2009). Histotechnology: A self-instructional text. American Society for Clinical Pathology Press, Hong Kong.

Cheng K. (2013). “Zebrafish atlas.” 2014, from

Cnudde V, Boone M, Dewanckele J, Dierick M, Van Hoorebeke L & Jacobs P (2011). 3d characterization of sandstone by means of x-ray computed tomography. Geosphere 7(1): 54-61.

Condon JC, Jeyasuria P, Faust JM & Mendelson CR (2004). Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition. PNAS 101(14): 4978-4983.

Cox PG & Jeffery N (2011). Reviewing the morphology of the jaw-closing musculature in squirrels, rats, and guinea pigs with contrast-enhanced microct. Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology 294(10): 915-928.

Degenhardt K, Wright AC, Horng D, Padmanabhan A & Epstein JA (2010). Rapid 3d phenotyping of cardiovascular development in mouse embryos by micro-ct with iodine staining. Circulation: Cardiovascular Imaging 3(3): 314-322.

Descamps E, Buytaert J, De Kegel B, Dirckx J & Adriaens D (2012). A qualitative comparison of 3d visualization in xenopus laevis using a traditional method and a non-destructive method. Belgian Journal of Zoology 142(2): 101-113.

Di Scipio F, Raimondo S, Tos P & Geuna S (2008). A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation. Microscopy Research and Technique 71(7): 497-502.

Dierick M, Van Loo D, Masschaele B, Boone M & Van Hoorebeke L (2010). A labview® based generic ct scanner control software platform. Journal of X-Ray Science and Technology 18(4): 451-461.

Dobrivojevic M, Bohacek I, Erjavec I, Gorup D & Gajovic S (2013). Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents. Croatian Medical Journal 54(1): 3-11.

Efimova OI, Khlebnikov AS, Senin RA, Voronin PA & Anokhin KV (2013). Contrasting of biological samples for x-ray synchrotron microtomography. Bulletin of Experimental Biology and Medicine 155(4): 413-416.

Fishman EK, Ney DR, Heath DG, Corl FM, Horton KM & Johnson PT (2006). Volume rendering versus maximum intensity projection in ct angiography: What works best, when, and why1. Radiographics 26(3): 905-922.

Gignac PM & Kley NJ (2014). Iodine-enhanced micro-ct imaging: Methodological refinements for the study of the soft-tissue anatomy of post-embryonic vertebrates. J Exp Zool B Mol Dev Evol 322(3): 166-176.

Golding RE & Jones AS (2007). Micro-ct as a novel technique for 3d reconstruction of molluscan anatomy. Molluscan Research 27: 123-128.

Hall,C, Jakus M & Schmitt F (1945). The structure of certain muscle fibrils as revealed by the use of electron stains. Journal of Applied Physics 16: 459-465.

Hardy RJ & Friedrich VL, Jr. (1996). Oligondendrocyte progenitors are generated throughout the embryonic mouse brain, but differentiate in restricted foci. Development 122: 2059-2069.

Hayat MA (2000). Principles and techniques of electron microscopy: Biological applications. Cambridge University Press. p.564.

Isogai S, Horiguchi M & Weinstein BM (2001). The vascular anatomy of the developing zebrafish: An atlas of embryonic and early larval development. Developmental Biology 230(2): 278-301.

Jeffery NS, Stephenson RS, Gallagher JA, Jarvis JC & Cox PG (2011). Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres. J Biomech 44(1): 189-192.

Johnson JT, Hansen MS, Wu I, Healy LJ, Johnson CR, Jones GM, Capecchi MR & Keller C (2006). Virtual histology of transgenic mouse embryos for high-throughput phenotyping. Plos Genetics 2(4): 471-477.

Jones AS, Milthorpe BK & Howlett CR (1994). Measurement of microtomy induced section distortion and its correction for 3-dimensional histological reconstructions. Cytometry 15: 95-105.

Keggin J (1934). The structure and formula of 12-phosphotungstic acid. Proceedings of the Royal Society of London. Series A 144(851): 75-100.

Kiernan JA (1981). Histological and histochemical methods. Theory and practice. Pergamon Press, Oxford.

Knudson CB & Knudson W (2001). Cartilage proteoglycans. Cell & Development Biology, Elsevier.

Lecker DN, Kumari S & Khan A (1997). Iodine binding capacity and iodine binding energy of glycogen. Journal of Polymer Science Part A: Polymer Chemistry 35(8): 1409-1412.

Ma S, Charron J & Erikson RL (2003). Role of plk2 (snk) in mouse development and cell proliferation. Molecular and Cellular Biology 23(19): 6936-6943.

Masschaele BC, Cnudde V, Dierick M, Jacobs P, Van Hoorebeke L & Vlassenbroeck J (2007). Ugct: New x-ray radiography and tomography facility. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 580(1): 266-269.

Metscher BD (2009a). Microct for comparative morphology: Simple staining methods allow high-contrast 3d imaging of diverse non-mineralized animal tissues. BMC Physiol 9: 11.

Metscher BD (2009b). Microct for developmental biology: A versatile tool for high-contrast 3d imaging at histological resolutions. Developmental Dynamics 238(3): 632-640.

Mizutani R & Suzuki Y (2012). X-ray microtomography in biology. Micron 43(2-3): 104-115.

Mizutani R, Takeuchi A, Uesugi K, Takekoshi S, Osamura RY & Suzuki Y (2008). X-ray microtomographic imaging of three-dimensional structure of soft tissues. Tissue Engineering Part C-Methods 14(4): 359-363.

Naveh GR, Brumfeld V, Dean M, Shahar R & Weiner S (2014). Direct microct imaging of non-mineralized connective tissues at high resolution. Connect Tissue Res 55(1): 52-60.

Patton JT & Kaufman MH (1995). The timing of ossification of the limb bones, and growth-rates of various long bones of the fore and hind limbs of the prenatal and early postnatal laboratory mouse. Journal of Anatomy 186: 175-185.

Pauwels E, Van Loo D, Cornillie P, Brabant L & Van Hoorebeke L (2013). An exploratory study of contrast agents for soft tissue visualization by means of high resolution x-ray computed tomography imaging. Journal of Microscopy 250(1): 21-31.

Pohlmann A, Möller M, Decker H & Schreiber WG (2007). Mri of tarantulas: Morphological and perfusion imaging. Magnetic Resonance Imaging 25(1): 129-135.

Quintarelli G, Zito R & Cifonelli J (1971). On phosphotungstic acid staining. I. Journal of Histochemistry & Cytochemistry 19(11): 641-647.

Ritman EL (2004). Micro-computed tomography-current status and developments. Annual Review of Biomedical Engineering 6: 185-208.

Ritman EL (2011). Current status of developments and applications of micro-ct. Annual Review of Biomedical Engineering, Vol 13 13: 531-552.

Roy MC, Nakanishi H, Takahashi K, Nakanishi S, Kajihara S, Hayasaka T, Setou M, Ogawa K, Taguchi R & Naito T (2011). Salamander retina phospholipids and their localization by maldi imaging mass spectrometry at cellular size resolution. Journal of Lipid Research 52(3): 463-470.

Rubin GD, Dake MD, Napel S, Jeffrey RB, McDonnell CH, Sommer FG, Wexler L & Williams DM (1994). Spiral ct of renal artery stenosis: Comparison of three-dimensional rendering techniques. Radiology 190(1): 181-189.

Santi PA (2011). Light sheet fluorescence microscopy: A review. Journal of Histochemistry & Cytochemistry 59(2): 129-138.

Schambach SJ, Bag S, Schilling L, Groden C & Brockmann MA (2010). Application of micro-ct in small animal imaging. Methods 50(1): 2-13.

Schambra U (2008). Prenatal mouse brain atlas. Springer Science+Business Media, LLC, New York.

Seki T, Sawamoto K, Parent JM & Alvarez-Buylla A (2011). Neurogenesis in the adult brain in Neurobiology. Springer, Tokyo.

Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sorensen J, Baldock R & Davidson D (2002). Optical projection tomography as a tool for 3d microscopy and gene expression studies. Science 296(5567): 541-545.

Stockwell RA (1979). Biology of cartilage cells. Cambridge University Press.

Tsai HP & Holliday CM (2011). Ontogeny of the alligator cartilago transiliens and its significance for sauropsid jaw muscle evolution. Plos One 6(9):e24935.

Tucker AS (2007). Salivary gland development. Seminars in Cell & Developmental Biology 18(2): 237-244.

Tyszka JM, Fraser SE & Jacobs RE (2005). Magnetic resonance microscopy: Recent advances and applications. Current Opinion in Biotechnology 16(1): 93-99.

Ullmann JF, Cowin G, Kurniawan ND & Collin SP (2010). A three-dimensional digital atlas of the zebrafish brain. Neuroimage 51(1): 76-82.

Vickerton P, Jarvis J & Jeffery N (2013). Concentration-dependent specimen shrinkage in iodine-enhanced microct. J Anat 223(2): 185-193.

Vlassenbroeck J, Dierick M, Masschaele B, Cnudde V, Van Hoorebeke L & Jacobs P (2007). Software tools for quantification of x-ray microtomography. Nuclear Instruments & Methods in Physics Research Section a-Accelerators Spectrometers Detectors and Associated Equipment 580(1): 442-445.

Warburton D, El-Hashash A, Carraro G, Tiozzo C, Sala F, Rogers O, De Langhe S, Kemp PJ, Riccardi D, Torday J, Bellusci S, Shi W, Lubkin SR & Jesudason E (2010). Lung organogenesis. Organogenesis in Development 90: 73-158.

Watling C, Lago N, Benmerah S, FitzGerald J, Tarte E, McMahon S, Lacour S & Cameron R (2010). Novel use of x-ray micro computed tomography to image rat sciatic nerve and integration into scaffold. Journal of Neuroscience Methods 188(1): 39-44.

Watson ML (1958). Staining of tissue sections for electron microscopy with heavy metals. The Journal of Biophysical and Biochemical Cytology 4(4): 475-478.

Weber KT, Sun Y, Tyagi SC & Cleutjens JPM (1994). Collagen network of the myocardium: Function, structural remodeling and regulatory mechanisms. Journal of Molecular and Cellular Cardiology 26(3): 279-292.

Wiechmann AF & Wirsig-Wiechmann CR (2003). Color atlas of xenopus laevis histology. Kluwer Academic Publishers, Boston.

Wigglesworth V (1975). Lipid staining for the electron microscope: A new method. Journal of Cell Science 19(3): 425-437.

Wilhelm G, Handschuh S, Plant J & Nemeschkal HLEO (2011). Sexual dimorphism in head structures of the weevil rhopalapion longirostre (olivier 1807) (coleoptera: Curculionoidea): A response to ecological demands of egg deposition. Biological Journal of the Linnean Society 104(3): 642-660.

Wirsén C & Larsson KS (1964). Histochemical differentiation of skeletal muscle in foetal and newborn mice. Journal of Embryology and Experimental Morphology 12: 758-767.

Wullimann MF, Rupp B & Reichert H (1996). Neuroanatomy of the zebrafish brain: A topological atlas. Birkhäuser Verlag, Basel.

Zanette I, Daghfous G, Weitkamp T, Gillet B, Adriaens D, Langer M, Cloetens P, Helfen L, Bravin A, Peyrin F, Baumbach T, Dischler J-M, Van Loo D, Praet T, Poirier-Quinot M & Boistel R (2013). Looking inside marine organisms with mri and x-ray tomography. In: Reynaud EG (Eds.). Imaging marine life. Wiley & Sons: 123-186.



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