Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools

An Erratum to this article was published on 25 September 2014

This article has been updated

Abstract

This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3–6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis.

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Figure 1: Experimental flowchart of key steps describing the three main parts of the protocol.
Figure 2: Schematic of the method used to hold mouse embryos for DSLM imaging.
Figure 3: Embryo transfer method.
Figure 4: Photograph of the assembled system around the chamber (left) and humidifier, gas mixture and temperature controller (right).
Figure 5: Graphical user interface of the manual tracking program.
Figure 6: Graphical user interface of the program for nuclei center extraction before semiautomatic tracking.
Figure 7: Graphical user interface of the semiautomatic tracking program.
Figure 8: Example of live imaging of whole mouse embryos at embryonic day 6.5.
Figure 9: Example of tracking analyses for epiblast and mesodermal nuclei.

Change history

  • 18 April 2014

     In the version of this article initially published, Table 1 contained a number of errors: lateral resolution for two-photon was erroneously stated as ‘r divided by the square root of 2’ instead of ‘r multiplied by the square root of 2’; the illumination intensity for two-photon microscopy was given as ‘103 times E’ instead of ‘106 times E’; ‘lateral resolution’ was assigned the incorrect footnote; and reference 11 was incorrectly cited as a source for the table. An additional footnote (a) has also been added to the version that was originally published. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank T. Fujimori for Histone H2B-GFP, H2B-mCherry and Lyn-Venus transgenic mice; H. Tao for handling of the mice; and F. Härle for DSLM software. We also thank members of the S.N. laboratory, the N. Ueno laboratory and the N. Shiina laboratory for valuable discussions, comments and technical assistance. We thank N. Papas for comments on the manuscript. This work was supported by a Grant-in-Aid for Young Scientists (A) from the Japan Society for the Promotion of Science (JSPS, 18687902); by the JSPS Fellows to T.I. (22353); by the Ministry of Education, Culture, Sports, Science and Technology (MEXT, 10J00353); by the Core Research for Evolutional Science and Technology (CREST) program; and by the Human Frontier Science Program (HFSP).

Author information

Affiliations

Authors

Contributions

T.I. developed most of the protocols described here and performed the experiments with the help of H.K.-K. under the supervision of S.N. K.N. developed the analysis tools under the supervision of A.M. P.J.K. and E.H.K.S. developed DSLM. T.I. and P.J.K. wrote the paper.

Corresponding authors

Correspondence to Takehiko Ichikawa or Shigenori Nonaka.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Effect of phototoxicity on mouse embryo during gastrulation using long wavelength illumination.

H2B–mCherry transgenetic mouse at embryonic day 6.5 was used. The embryo was illuminated with 647 nm laser light in 100 optical sections per image stack, once every 5 minutes. Continuous illumination with long wavelength light for 12 hours does not cause embryo degradation. Scale bar: 20 μm.

Supplementary Figure 2 Design drawing of embryo holder.

The holder is made from a 5-mm-diameter acrylic rod. The left side of the holder is glued to the piston of a 1-ml syringe.

Supplementary Figure 3 Design drawing of gas adapter.

The adapter is made from a 2-mm-thick acrylic plate.

Supplementary Figure 4 Digital scanned light-sheet microscope setup and light path.

Laser beam from argon-krypton laser is selected with AOTF. A light sheet is formed using the two-axis high-speed scan head and f-theta lens. The light sheet excites fluorescent molecules in the sample. Fluorescence is detected via the detection lens, long pass filter and CCD camera.

Supplementary Figure 5 Examples of optical sections from live imaging experiments with mouse embryos at embryonic day (E) 5.5 to 7.5.

Top shows nuclear stained embryo (H2B–mCherry) and bottom shows membrane stained embryo (Lyn–Venus). Scale bar = 20 μm.

Supplementary information

Supplementary Figure 1

Effect of phototoxicity on mouse embryo during gastrulation using long wavelength illumination. (PDF 155 kb)

Supplementary Figure 2

Design drawing of embryo holder. (PDF 87 kb)

Supplementary Figure 3

Design drawing of gas adapter. (PDF 82 kb)

Supplementary Figure 4

Digital scanned light-sheet microscope setup and light path. (PDF 232 kb)

Supplementary Figure 5

Examples of optical sections from live imaging experiments with mouse embryos at embryonic day (E) 5.5 to 7.5. (PDF 248 kb)

Live imaging of Histone H2B–mCherry mouse embryo at embryonic day 6.5.

The optical section shown here is located about 85 μm from the distal end of the embryo. The time interval is 5 min. (MOV 4722 kb)

Supplementary Data 1

Programs described in this protocol. The archive includes Matlab scripts for image projection and cropping, the ImageJ macro for image registration, compiled programs for manual and semi-automatic tracking and the POV-Ray script for visualizing trajectories. Descriptions of each program are provided in Table 2. (ZIP 230 kb)

Supplementary Data 2

Source code of manual and semi-automatic tracking programs. The archive includes source code for “manual_tracker.exe”, “cdots_search.exe”, “ichi_srcx.exe”, “semiauto_tracer.exe”, “cc_trace.exe” and “cc_reduce.exe”. These programs were written in C++ and C# using Visual Studio 2010 (Microsoft). (ZIP 374 kb)

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Ichikawa, T., Nakazato, K., Keller, P. et al. Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools. Nat Protoc 9, 575–585 (2014). https://doi.org/10.1038/nprot.2014.035

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