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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.
Figure 1: Experimental setup for propagation-based phase-contrast X-ray microtomography.
Figure 7: Horizontal slice through reconstructed volume for high count rates.
Figure 11: Quantitative assessment of morphological changes. (a,b) 3D renderings of cavities within the PS between ARC and BLC for times 62 and 114 min. (c) Volume change of ARC, BLC and gastrula from 52 to 114 min.
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