Hostname: page-component-cd9895bd7-q99xh Total loading time: 0 Render date: 2024-12-27T08:23:21.310Z Has data issue: false hasContentIssue false
  • English
  • Français

Phase Contrast Synchrotron Microtomography: Improving Noninvasive Investigations of Fossil Embryos In Ovo

Published online by Cambridge University Press:  18 January 2012

Vincent Fernandez ,
Eric Buffetaut ,
Eric Maire ,
Jérôme Adrien ,
Varavudh Suteethorn  and
Paul Tafforeau
Vincent Fernandez*
Affiliation:
European Synchrotron Radiation Facility, X-Ray Imaging Group, 6 rue Horowitz BP 220, 38046 Grenoble Cedex, France
Eric Buffetaut
Affiliation:
CNRS (UMR 8538), Laboratoire de Géologie de l'École Normale Supérieure, 24 rue Lhomond, 75231 Paris Cedex 05, France
Eric Maire
Affiliation:
Université de Lyon, INSA-Lyon, MATEIS CNRS (UMR 5510), 25 avenue jean Capelle, 69621, Villeurbanne, France
Jérôme Adrien
Affiliation:
Université de Lyon, INSA-Lyon, MATEIS CNRS (UMR 5510), 25 avenue jean Capelle, 69621, Villeurbanne, France
Varavudh Suteethorn
Affiliation:
Bureau of Fossil Research and Museum, Department of Mineral Resources, Rama VI Road, Bangkok 10400, Thailand
Paul Tafforeau
Affiliation:
European Synchrotron Radiation Facility, X-Ray Imaging Group, 6 rue Horowitz BP 220, 38046 Grenoble Cedex, France
*
Corresponding author. E-mail: Vincent.Fernandez@wits.ac.za
Get access

Abstract

Fossil embryos are paramount for our understanding of the development of extinct species. However, although thousands of fossil amniote eggs are known, very few embryos in ovo have been described. First reports of fossil embryos were based on broken eggs, where the embryonic remains were already exposed, because destructive methods on complete eggs were avoided. Investigations of complete eggs therefore required nondestructive approaches, such as X-ray microtomography (μCT). However, due to the general low density contrast between fossilized bones and infilling matrix, only a few specimens have been reported using these techniques. Using propagation phase contrast X-ray synchrotron microtomography (PPC-SR-μCT), we report here the discovery of three well-preserved embryos in Early Cretaceous eggs from Thailand. By scanning these eggs using different imaging techniques, we show that vastly different interpretations can be made regarding the preservation state and/or the developmental stage of these embryos. PPC-SR-μCT also revealed differential contrast between bone categories, presumably reflecting the ossification pattern of these embryos. Applying such an approach to large-scale studies of fossil eggs could lead to more discoveries and detailed studies of fossil embryos, providing important developmental and phylogenetic information on extinct and extant amniotes.

Keywords

phase contrastmicrotomographyfossil eggfossil embryo
Type
Techniques Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 1 , February 2012 , pp. 179 - 185
Copyright
Copyright © Microscopy Society of America 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

Current address: Bernard Price Institute for Palaeontological Research, University of the Witwatersrand, Private Bag 3, Johannesburg 2050, South Africa

References

REFERENCES

Allman, B.E., McMahon, P.J., Nugent, K.A., Paganin, D., Jacobson, D.L., Arif, M. & Werner, S.A. (2000). Imaging: Phase radiography with neutrons. Nature 408, 158–159.CrossRefGoogle Scholar
Balanoff, A.M., Norell, M.A., Grellet-Tinner, G. & Lewin, M.R. (2008). Digital preparation of a probable neoceratopsian preserved within an egg, with comments on microstructural anatomy of ornithischian eggshells. Naturwissenschaften 95, 493–500.CrossRefGoogle ScholarPubMed
Balanoff, A.M. & Rowe, T. (2007). Osteological description of an embryonic skeleton of the extinct elephant bird, Aepyornis (Palaeognathae. Ratitae). J Vertebr Paleontol Memoir 9, 1–53.Google Scholar
Baruchel, J., Lodini, A., Romanzetti, S., Rustichelli, F. & Scrivani, A. (2001). Phase-contrast imaging of thin biomaterials. Biomaterials 22, 1515–1520.Google Scholar
Buffetaut, E., Grellet-Tinner, G., Suteethorn, V., Cuny, C., Tong, H., Kosir, A., Cavin, L., Chitsing, S., Griffiths, J., Tabouelle, J. & LeLoeuff, J. (2005). Minute theropod eggs and embryo from the Lower Cretaceous of Thailand and the dinosaur-bird transition. Naturwissenschaften 92, 477–482.CrossRefGoogle ScholarPubMed
Carlson, K.J., Stout, D., Jashashvili, T., de Ruiter, D.J., Tafforeau, P., Carlson, K. & Berger, L.R. (2011). The endocast of MH1, Australopithecus sediba. Science 333, 1402–1407.CrossRefGoogle ScholarPubMed
Carpenter, K. (1999). Eggs, Nests, & Baby Dinosaurs. Bloomington, IN: Indiana University Press.Google Scholar
Chiappe, L., Coria, R., Dingus, L., Jackson, F., Chinsamy, A. & Fox, M. (1998). Sauropod dinosaur embryos from the Late Cretaceous of Patagonia. Nature 396(6708), 258–261.Google Scholar
Cloetens, P., Barrett, R., Baruchel, J., Guigay, J.-P. & Schlenker, M. (1996). Phase objects in synchrotron radiation hard X-ray imaging. J Phys D: Appl Phys 29, 133–146.Google Scholar
Delfino, M. & Sánchez-Villagra, M.R. (2010). A survey of the rock record of reptilian ontogeny. Semin Cell Biol 21, 432–440.Google Scholar
Dierick, M., Cnudde, V., Masschaele, B., Vlassenbroeck, J., Van Hoorebeke, L. & Jacobs, P. (2007). Micro-CT of fossils preserved in amber. Nucl Instrum Meth A 580, 641–643.CrossRefGoogle Scholar
Domanus, J.C. (1992). Practical Neutron Radiograph. New York: Springer.CrossRefGoogle Scholar
Fernandez, V. (2010). Detection and imaging of fossilized in ovo embryos using synchrotron based microtomography: Study of the enigmatic embryos from Phu Phok (Lower Cretaceous, Thailand). PhD Thesis, University Pierre et Marie Curie Paris VI.Google Scholar
Grellet-Tinner, G., Chiappe, L., Norell, M.A. & Bottjer, D. (2006). Paleobiology of dinosaur eggs & nesting behaviors. Palaeogeogr Palaeoclimatol Palaeoecol 232, 294–321.Google Scholar
Grellet-Tinner, G., Sim, C.M., Kim, D.H., Trimby, P., Higa, A., An, S.L., Oh, H.S., Kim, T.J. & Kardjilov, N. (2011). Description of the first lithostrotian titanosaur embryo in ovo with Neutron characterization and implications for lithostrotian Aptian migration and dispersion. Gondwana Res 20, 621–629.Google Scholar
Grigorescu, D., Garcia, G., Csiki, Z., Codrea, V. & Bojar, A.-V. (2010). Uppermost Cretaceous megaloolithid eggs from the Haţeg Bassin, Romania, associated with hadrosaur hatchling: Search for explanation. Palaeogeogr Palaeoclimatol Palaeoecol 293, 360–374.Google Scholar
Grigorescu, D., Weishampel, D., Norman, D., Seclamen, M., Rusu, M., Baltres, A. & Teodorscu, V. (1994). Late Maastrichtian dinosaur eggs from the Haţeg Bassin (Romania). In Dinosaur Eggs and Babies, Carpenter, K., Hirsh, K.F. & Horner, J.R. (Eds.), pp. 75–87. New York: Cambridge University Press.Google Scholar
Holden, C. (1994). Digging out a dinosaur embryo. Science 263(5146), 469.Google Scholar
Horner, J.R. & Weishampel, D.B. (1996). Correction to a comparative embryological study of two ornithischian dinosaurs (1988). Nature 383, 103.CrossRefGoogle Scholar
Kardjilov, N., Lehmann, E., Steichele, E. & Vontobel, P. (2004). Phase-contrast radiography with a polychromatic neutron beam. Nucl Instrum Methods Phys Res A 527, 519–530.CrossRefGoogle Scholar
Kottler, C., Pfeiffer, F., Bunk, O., Grünzweig, C., Bruder, J., Kaufmann, R., Tlustos, L., Walt, H., Briod, I., Weitkamp, T. & David, C. (2007). Phase contrast X-ray imaging of large samples using an incoherent laboratory source. Phys Status Solidi A 204(8), 2728–2733.CrossRefGoogle Scholar
Kundrát, M., Cruickshank, A.R.I., Manning, T.W. & Nudds, J. (2008). Embryos of therizinosauroid theropods from the Upper Cretaceous of China. Diagnosis and analysis of ossification patterns. Acta Zool (Stockholm) 89, 231–251.Google Scholar
Lak, M., Néraudeau, D., Nel, A., Cloetens, P., Perrichot, V. & Tafforeau, P. (2008). Phase contrast X-ray synchrotron imaging: Opening access to fossil inclusions in opaque amber. Microsc Microanal 14, 251–259.CrossRefGoogle ScholarPubMed
Liston, J.J. & McJury, M. (2003). Egg candling for the 21st century: The use of three dimensional digital imaging technology to investigate the contens of fossilised eggs. Q J Dinosaur Soc 4(4), 6–9.Google Scholar
McMahon, P.J., Allman, B.E., Jacobson, D.L., Arif, M., Werner, S.A. & Nugent, K.A. (2003). Quantitative phase radiography with polychromatic neutrons. Phys Rev Lett 91(14), 145502(4).Google Scholar
Norell, M.A., Clark, J.M., Demberelyin, D., Rhinchen, B., Chiappe, L.M., Davidson, A.R., McKenna, M.C., Altangerel, P. & Novacek, M.J. (1994). A theropod dinosaur embryo and the affinities of the Flaming Cliffs dinosaur eggs. Science 266, 779–782.Google Scholar
Olejniczak, A.J., Tafforeau, P., Feeney, R.N.M. & Martin, L.B. (2008). Three-dimensional primate molar enamel thickness. J Hum Evol 54, 187–195.CrossRefGoogle ScholarPubMed
Pfeiffer, F., Grünzweig, C., Bunk, O., Frei, G., Lehmann, E. & David, C. (2006a). Neutron phase imaging and tomography. Phys Rev Lett 96, 215505(4).Google Scholar
Pfeiffer, F., Kottler, C., Bunk, O. & David, C. (2007). Hard X-ray phase tomography with low-brilliance sources. Phys Rev Lett 98, 108105(4).CrossRefGoogle ScholarPubMed
Pfeiffer, F., Weitkamp, T., Bunk, O. & David, C. (2006b). Phase retrieval and differential phase-contrast imaging with low-brilliance X-ray sources. Nat Phys 2, 258–261.Google Scholar
Remes, K., Huang, T.D., Ruf, I., Zhong, S. & Yang, C. (2009). Discovery of an embryonic basal sauropodomorph in the Lower Jurassic Lufeng Formation, People's Republic of China. In Fourth International Symposium on Dinosaur Eggs and Babies, Varricchio, D.J. & Jackson, F.J. (Eds). Bozeman, MT: Montana State University.Google Scholar
Schwarz, D., Vontobel, P., Lehmann, E.H., Meyer, C.A. & Bongartz, G. (2005). Neutron tomography of internal structures of vertebrate remains: A comparison with X-ray computed tomography. Palaeontol Electron 8, 30A, 11p.Google Scholar
Smith, T.M., Olejniczak, A.J., Kupczik, K., Lazzari, V., de Vos, J., Kullmer, O., Schrenk, F., Hublin, J.-J., Jacob, T. & Tafforeau, P. (2009). Taxonomic assessment of the trinil molars using nondestructive 3D structural and development analysis. PaleoAnthropol 2009, 117–129.Google Scholar
Smith, T.M. & Tafforeau, P. (2008). New visions of dental tissue research. Tooth development, chemistry and structure. Evol Anthropol 17, 213–226.CrossRefGoogle Scholar
Snigirev, A., Kohn, V., Snigireva, I. & Lengeler, B. (1996). A compound refractive lens for focusing high-energy X-rays. Nature 384, 49–51.CrossRefGoogle Scholar
Strobl, M., Manke, I., Kardjilov, N., Hilger, A., Dawson, M. & Banhart, J. (2009). Advances in neutron radiography and tomography. J Phys D: Appl Phys 42, 1–21.Google Scholar
Strobl, M., Staack, K., Treimer, W. & Hilger, A. (2008a). Quantitative neutron phase contrast tomography. Meas Sci Technol 19, 1–4.CrossRefGoogle Scholar
Strobl, M., Treimer, W., Kardjilov, N., Hilger, A. & Zabler, S. (2008b). On neutron phase contrast imaging. Nucl Instrum Methods Phys Res B 266, 181–186.Google Scholar
Sutton, M.D. (2008). Tomographic techniques for the study of exceptionally preserved fossils. Proc R Soc London Ser B 275, 1–7.Google Scholar
Tafforeau, P., Boistel, R., Boller, E., Bravin, A., Brunet, M., Chaimanee, Y., Cloetens, P., Feist, M., Hoszowska, J., Jaeger, J.-J., Kay, R., Lazzari, V., Marivaux, L., Nel, A., Nemoz, C., Thibault, X., Vignaud, P. & Zabler, S. (2006). Applications of X-ray synchrotron microtomography for non-destructive 3D studies of paleontological specimens. Appl Phys A 83, 195–202.CrossRefGoogle Scholar
Varricchio, D., Horner, J. & Jackson, F. (2002). Embryos and eggs for the Cretaceous theropod dinosaur Troodon formosus. J Vertebr Paleontol 22 (3), 564–576.Google Scholar
Varricchio, D., Jackson, F., Borkowski, J. & Horner, J. (1997). Nest and egg clutches of the dinosaur Troodon formosus and the evolution of avian reproductive traits. Nature 385(6613), 247–250.Google Scholar
Vontobel, P., Lehmann, E. & Carlson, W.D. (2005). Comparison of X-ray and neutron tomography—Investigations of geological materials. IEEE Trans Nucl Sci 52, 338–341.CrossRefGoogle Scholar
Weitkamp, T., Zanette, I., David, C., Baruchel, J., Bech, M., Bernard, P., Deyhle, H., Donath, T., Kenntner, J., Lang, S., Mohr, J., Müller, B., Pfeiffer, F., Reznikova, E., Rutishauser, S., Schulz, G., Tapfer, A. & Valade, J.P. (2010). Recent developments in X-ray Talbot interferometry at ESRF-ID19. Proc SPIE 7804, 780406-(5).Google Scholar
Wilkins, S.W., Gureyev, T.E., Gao, D., Pogany, A. & Stevenson, A.W. (1996). Phase-contrast imaging using polychromatic hard X-rays. Nature 384, 335–338.Google Scholar
Winkler, B. (2006). Applications of neutron radiography and neutron tomography. Rev Mineral Geochem 63, 459–471.Google Scholar
Witzmann, F., Scholz, H., Müller, J. & Kardjilov, N. (2010). Sculpture and vascularization of dermal bones, and the implications for the physiology of basal tetrapods. Zool J Linn Soc 160, 302–340.CrossRefGoogle Scholar
Supplementary material: PDF

Fernandez et al. supplementary movie

Supplementary figure

Download Fernandez et al. supplementary movie(PDF)
PDF 187 KB

Fernandez et al. supplementary movie

Movie 1

Download Fernandez et al. supplementary movie(Video)
Video 12.2 MB