%0 Journal Article %1 moosmann2014timelapse %A Moosmann, Julian %A Ershov, Alexey %A Weinhardt, Venera %A Baumbach, Tilo %A Prasad, Maneeshi S %A LaBonne, Carole %A Xiao, Xianghui %A Kashef, Jubin %A Hofmann, Ralf %D 2014 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat. Protocols %K biology embryos in-vivo motion_analysis myPUB tomography xenopus %N 2 %P 294--304 %T Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis %U http://dx.doi.org/10.1038/nprot.2014.033 %V 9 %X 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.

@article{moosmann2014timelapse, 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.}, added-at = {2014-01-14T09:15:30.000+0100}, author = {Moosmann, Julian and Ershov, Alexey and Weinhardt, Venera and Baumbach, Tilo and Prasad, Maneeshi S and LaBonne, Carole and Xiao, Xianghui and Kashef, Jubin and Hofmann, Ralf}, biburl = {https://www.bibsonomy.org/bibtex/2a5789cce25f004fa98731b2b4c42bff5/alex_ruff}, description = {Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis : Nature Protocols : Nature Publishing Group}, interhash = {b235f9ac8afc12c86d379ce17c779366}, intrahash = {a5789cce25f004fa98731b2b4c42bff5}, issn = {17542189}, journal = {Nat. Protocols}, keywords = {biology embryos in-vivo motion_analysis myPUB tomography xenopus}, month = feb, number = 2, pages = {294--304}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, timestamp = {2014-01-14T09:15:30.000+0100}, title = {Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis}, url = {http://dx.doi.org/10.1038/nprot.2014.033}, volume = 9, year = 2014 }