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

• Published in: Nature protocols Vol. 9; no. 2; pp. 294 - 304
• Main Authors: Moosmann, Julian, Ershov, Alexey, Weinhardt, Venera, Baumbach, Tilo, Prasad, Maneeshi S, LaBonne, Carole, Xiao, Xianghui, Kashef, Jubin, Hofmann, Ralf
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-02-2014; Nature Publishing Group
• Subjects: 631/114/1564; 631/136/1660; 631/136/334/1874/761; 631/1647/245/1847; Amphibians; Analytical Chemistry; Animals; Biological Techniques; Biologists; Biology; Cameras; Computational Biology/Bioinformatics; Cooperation; Data acquisition; Data analysis; Data interpretation; Data processing; Developmental stages; Electrons; Embryonic development; Embryos; Flux density; Gastrula - physiology; Gastrula - ultrastructure; Gastrulation; Gastrulation - physiology; Image contrast; Image processing; Image reconstruction; Image resolution; Image segmentation; Imaging, Three-Dimensional; Laboratories; Life Sciences; Mathematicians; Methods; Microarrays; Microscopy, Phase-Contrast - methods; Microtomography; Morphogenesis; Observations; Organic Chemistry; Phase retrieval; Physicists; Physiological aspects; Protocol; Radiation; Radiolysis; Sample preparation; Scientists; Sensors; Spatial discrimination; Spatial resolution; Time-Lapse Imaging - methods; Tomography; Vertebrates; X-Ray Microtomography - methods; X-rays; Xenopus; Xenopus laevis - embryology; France; United States > US; Illinois; Germany
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2014.033

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.