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.
| Original language | English |
|---|---|
| Pages (from-to) | 294-304 |
| Number of pages | 11 |
| Journal | Nature Protocols |
| Volume | 9 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 1 Feb 2014 |
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ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
Cite this
Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis. / Moosmann, Julian; Ershov, Alexey; Weinhardt, Venera; Baumbach, Tilo; Prasad, Maneeshi S.; Labonne, Carole; Xiao, Xianghui; Kashef, Jubin; Hofmann, Ralf.
In: Nature Protocols, Vol. 9, No. 2, 01.02.2014, p. 294-304.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis
AU - Moosmann, Julian
AU - Ershov, Alexey
AU - Weinhardt, Venera
AU - Baumbach, Tilo
AU - Prasad, Maneeshi S.
AU - Labonne, Carole
AU - Xiao, Xianghui
AU - Kashef, Jubin
AU - Hofmann, Ralf
PY - 2014/2/1
Y1 - 2014/2/1
N2 - 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.
AB - 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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U2 - 10.1038/nprot.2014.033
DO - 10.1038/nprot.2014.033
M3 - Article
C2 - 24407356
AN - SCOPUS:84893275927
VL - 9
SP - 294
EP - 304
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
IS - 2
ER -