Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis
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 l...
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| Hauptverfasser: | , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
09 January 2014
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| In: |
Nature protocols
Year: 2014, Jahrgang: 9, Heft: 2, Pages: 294-304 |
| ISSN: | 1750-2799 |
| DOI: | 10.1038/nprot.2014.033 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1038/nprot.2014.033 Verlag, lizenzpflichtig, Volltext: https://www.nature.com/articles/nprot.2014.033 
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| Verfasserangaben: | Julian Moosmann, Alexey Ershov, Venera Weinhardt, Tilo Baumbach, Maneeshi S. Prasad, Carole LaBonne, Xianghui Xiao, Jubin Kashef & Ralf Hofmann |
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| 520 | |a 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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