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Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis

Nature Protocols volume 9, pages 294–304 (2014)Cite this article

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Abstract

X-ray phase-contrast microtomography (XPCμT) is a label-free, high-resolution imaging modality for analyzing early development of vertebrate embryos in vivo by using time-lapse sequences of 3D volumes. Here we provide a detailed protocol for applying this technique to study gastrulation in Xenopus laevis (African clawed frog) embryos. In contrast to μMRI, XPCμT images optically opaque embryos with subminute temporal and micrometer-range spatial resolution. We describe sample preparation, culture and suspension of embryos, tomographic imaging with a typical duration of 2 h (gastrulation and neurulation stages), intricacies of image pre-processing, phase retrieval, tomographic reconstruction, segmentation and motion analysis. Moreover, we briefly discuss our present understanding of X-ray dose effects (heat load and radiolysis), and we outline how to optimize the experimental configuration with respect to X-ray energy, photon flux density, sample-detector distance, exposure time per tomographic projection, numbers of projections and time-lapse intervals. The protocol requires an interdisciplinary effort of developmental biologists for sample preparation and data interpretation, X-ray physicists for planning and performing the experiment and applied mathematicians/computer scientists/physicists for data processing and analysis. Sample preparation requires 9–48 h, depending on the stage of development to be studied. Data acquisition takes 2–3 h per tomographic time-lapse sequence. Data processing and analysis requires a further 2 weeks, depending on the availability of computing power and the amount of detail required to address a given scientific problem.

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Figure 1: Experimental setup for propagation-based phase-contrast X-ray microtomography.
Figure 2: Schematic drawing of sample holder preparation.
Figure 3: Correspondence between radiographs and reconstructed, central-horizontal slice.
Figure 4: Removal of a hot pixel.
Figure 5: Removal of stripe artifacts, flat-field and dark-field correction.
Figure 6: Pre-processed intensity maps and ring-artifact removal by sinogram filtering.
Figure 7: Horizontal slice through reconstructed volume for high count rates.
Figure 8: Horizontal slice through reconstructed volume for low count rates.
Figure 9: Structure and dynamics of X. laevis gastrulation.
Figure 10: Analysis of differential cell movement to distinguish active from passive and individual from collective behavior.
Figure 11: Quantitative assesment of morphological changes.

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Acknowledgements

We thank T. van de Kamp for his help visualizing the setup and the sample-holder preparation, as well as F. de Carlo for allocating beam time at 2-BM-B station of APS, Argonne National Laboratory. The use of the APS, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. J.K.'s Young Investigator Group received financial support from the 'Concept for the Future' program of Karlsruhe Institute of Technology within the framework of the German Excellence Initiative. This research partially was funded by the German Federal Ministry of Education and Research under grant nos. 05K12CK2 and 05K12VH1, as well as by COST action MP1207.

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Authors and Affiliations

  1. Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany

    Julian Moosmann, Alexey Ershov, Tilo Baumbach & Ralf Hofmann

  2. Department of General Physics, Institute of Physics and Technology, National Research Tomsk Polytechnic University, Tomsk, Russia

    Alexey Ershov

  3. Laboratory for Applications of Synchrotron Radiation, Karlsruhe Institute of Technology, Karlsruhe, Germany

    Venera Weinhardt & Tilo Baumbach

  4. Centre for Organismal Studies, Universität Heidelberg, Heidelberg, Germany

    Venera Weinhardt

  5. Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA

    Maneeshi S Prasad & Carole LaBonne

  6. Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, USA

    Xianghui Xiao

  7. Cell and Developmental Biology, Zoological Institute II, Karlsruhe Institute of Technology, Karlsruhe, Germany

    Jubin Kashef

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Contributions

R.H. drafted the manuscript, and J.M., J.K. and A.E. contributed text. Figures were prepared by J.M., A.E and J.K. V.W., T.B., C.L., M.S.P. and X.X. provided critical review. All authors contributed to the final manuscript.

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Correspondence to Jubin Kashef or Ralf Hofmann.

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Moosmann, J., Ershov, A., Weinhardt, V. et al. Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis. Nat Protoc 9, 294–304 (2014). https://doi.org/10.1038/nprot.2014.033

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