title: Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis.
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.
authors.affiliation: Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Moosmann
authors.affiliation: 1] Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany. [2] Department of General Physics, Institute of Physics and Technology, National Research Tomsk Polytechnic University, Tomsk, Russia.
authors.forename: Alexey
authors.initials: A
authors.lastname: Ershov
authors.affiliation: 1] Laboratory for Applications of Synchrotron Radiation, Karlsruhe Institute of Technology, Karlsruhe, Germany. [2] Centre for Organismal Studies, Universität Heidelberg, Heidelberg, Germany.
authors.forename: Venera
authors.initials: V
authors.lastname: Weinhardt
authors.affiliation: 1] Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany. [2] Laboratory for Applications of Synchrotron Radiation, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Tilo
authors.initials: T
authors.lastname: Baumbach
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.
authors.forename: Maneeshi S
authors.initials: MS
authors.lastname: Prasad
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.
authors.forename: Carole
authors.initials: C
authors.lastname: LaBonne
authors.affiliation: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, USA.
authors.forename: Xianghui
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Cell and Developmental Biology, Zoological Institute II, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Jubin
authors.initials: J
authors.lastname: Kashef
authors.affiliation: Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hofmann
mesh_terms: D000818:Animals; D005775:Gastrula / Q000502:physiology / Q000648:ultrastructure*; D054262:Gastrulation / Q000502:physiology*; D021621:Imaging, Three-Dimensional; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D059008:Time-Lapse Imaging / Q000379:methods*; D055114:X-Ray Microtomography / Q000379:methods*; D014982:Xenopus laevis / Q000196:embryology*