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High-resolution reconstruction of the beating zebrafish heart

Nature Methods volume 11, pages 919922 (2014) | Download Citation

Abstract

The heart′s continuous motion makes it difficult to capture high-resolution images of this organ in vivo. We developed tools based on high-speed selective plane illumination microscopy (SPIM), offering pristine views into the beating zebrafish heart. We captured three-dimensional cardiac dynamics with postacquisition synchronization of multiview movie stacks, obtained static high-resolution reconstructions by briefly stopping the heart with optogenetics and resolved nonperiodic phenomena by high-speed volume scanning with a liquid lens.

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References

  1. 1.

    Cardiovasc. Res. 91, 279–288 (2011).

  2. 2.

    et al. Circ. Res. 106, 1342–1350 (2010).

  3. 3.

    et al. Nat. Genet. 31, 106–110 (2002).

  4. 4.

    et al. Nature 421, 172–177 (2003).

  5. 5.

    ., , , & PLoS Biol. 5, e53 (2007).

  6. 6.

    , , , & Science 305, 1007–1009 (2004).

  7. 7.

    & Opt. Lett. 32, 2608–2610 (2007).

  8. 8.

    , , & Development 139, 3242–3247 (2012).

  9. 9.

    , , & Development 135, 1179–1187 (2008).

  10. 10.

    , , , & J. Biomed. Opt. 10, 054001 (2005).

  11. 11.

    et al. Development 141, 585–593 (2014).

  12. 12.

    et al. J. Biomed. Opt. 16, 116021 (2011).

  13. 13.

    , , , & IEEE Trans. Med. Imaging 32, 578–588 (2013).

  14. 14.

    et al. Nat. Methods 4, 311–313 (2007).

  15. 15.

    , , & Nat. Methods 7, 418–419 (2010).

  16. 16.

    Anat. Rec. 102, 289–298 (1948).

  17. 17.

    et al. Am. J. Physiol. Heart Circ. Physiol. 300, H879–H891 (2011).

  18. 18.

    et al. Development 137, 3867–3875 (2010).

  19. 19.

    , , & Science 330, 971–974 (2010).

  20. 20.

    , , , & Opt. Express 21, 21010–21026 (2013).

  21. 21.

    & Zebrafish (Oxford University Press, 2002).

  22. 22.

    et al. Nat. Immunol. 4, 1238–1246 (2003).

  23. 23.

    , , , & Development 132, 5199–5209 (2005).

  24. 24.

    , , , & Dev. Dyn. 228, 30–40 (2003).

  25. 25.

    et al. Genes Dev. 22, 734–739 (2008).

  26. 26.

    , & Proc. Natl. Acad. Sci. USA 106, 17968–17973 (2009).

  27. 27.

    , & Toxicol. Appl. Pharmacol. 193, 370–382 (2003).

  28. 28.

    et al. Nat. Methods 9, 676–682 (2012).

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Acknowledgements

This work was supported by the Max Planck Society, the Human Frontier Science Program (CDA 00063/2010-C to J.H.) and a fellowship to M.M. from the Boehringer Ingelheim Fonds. We thank H. Otsuna for assistance with FluoRender and A. Reade, T. Op't Hof, R. Coronel, D.Y.R. Stainier and members of the Huisken laboratory for their comments. Initial work on this project was performed by J.H. in the labs of E.H.K. Stelzer, J. Wittbrodt and D.Y.R. Stainier.

Author information

Author notes

    • Sonja Hombach

    Present address: Institute of Biochemistry, Genetics and Microbiology, University of Regensburg, Regensburg, Germany.

Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

    • Michaela Mickoleit
    • , Benjamin Schmid
    • , Michael Weber
    • , Florian O Fahrbach
    • , Sonja Hombach
    •  & Jan Huisken
  2. Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

    • Sven Reischauer
  3. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA.

    • Sven Reischauer

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Contributions

M.M. and M.W. designed and built the SPIM setup. M.M. developed the single-view synchronization routine, performed and analyzed all experiments and visualized the data. B.S. developed the dual-view synchronization algorithm as well as the synthetic-heart-tube model and wrote the software to operate the SPIM setup. M.W. built the hardware for optogenetic manipulation. M.W. and M.M. performed the optogenetic experiments. F.O.F. designed, built, programmed and operated the ETL-SPIM setup. S.H. made the Tg(myl7:Gal4) line, and S.R. made the Tg(myl7:lifeactGFP) line. J.H. designed and supervised the project. M.M. and J.H. wrote the manuscript with contributions by M.W., B.S. and F.O.F.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jan Huisken.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

Videos

  1. 1.

    Shape of the beating heart at various contraction phases.

    Image of a 48 h.p.f. Tg(myl7:GFP) embryo with segmented outlines (pink dotted lines) of the heart over a full cardiac cycle.

  2. 2.

    Movie stack synchronization.

    Three synchronized planes of a movie stack of a 48 h.p.f. Tg(myl7:GFP) embryo and the 3D reconstruction. Scale bar 30 μm.

  3. 3.

    Single view synchronization yields almost isotropic resolution.

    Synchronized movie stack of a 48 h.p.f. Tg(myl7:GFP) embryo in maximum projected front view and a horizontal and vertical cut. Scale bar 30 μm.

  4. 4.

    Dual view sync of synthetic movie stacks.

    Reconstructions from movie stacks of a synthetic heart tube model synchronized using single- or dual-view synchronization. Scale bar 30 μm.

  5. 5.

    Dual view sync of real movie stacks.

    Reconstructions from movie stacks of a 30 h.p.f. Tg(myl7:GFP) embryo synchronized using single- or dual-view synchronization. Scale bar 30 μm.

  6. 6.

    Reconstruction of endo- and myocardium in a 30 h.p.f. embryo.

    Maximum projections showing cardiac contraction in real speed and slow motion in a 30 h.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo. Scale bar 30 μm.

  7. 7.

    Reconstruction of endo- and myocardium in a 48 h.p.f. embryo.

    Volume rendering (left) and single slice (right) showing cardiac cycle in real speed and slow motion in a 48 h.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo. Scale bar 30 μm.

  8. 8.

    Reconstruction of endo- and myocardium in a 72 h.p.f. embryo.

    Volume rendering of three different views showing cardiac contractions in real speed and slow motion in a 72 h.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo.

  9. 9.

    Reconstruction of endo- and myocardium in a 5 d.p.f. embryo.

    Single slices in front and side view showing cardiac contractions in a 5 d.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo.

  10. 10.

    Optogenetically stopped heart.

    Volume rendering showing the optogenetically stopped heart of a 5 d.p.f. Tg(myl7:lifeactGFP, myl7:Gal4, UAS:NpHR-mCherry) embryo.

  11. 11.

    4D reconstruction of blood flow with single view synchronization.

    Front and side view of synchronized heart of a 2 d.p.f. Tg(gata1a:DsRed, myl7:GFP) embryo. Scale bar 30 μm.

  12. 12.

    4D reconstruction of blood flow with ETL-SPIM.

    Front and side view of synchronized heart of a 2 d.p.f. Tg(gata1a:DsRed, myl7:GFP) embryo. Scale bar 30 μm.

  13. 13.

    Arrhythmic heart imaged with ETL-SPIM.

    Maximum projection of heart in 55 h.p.f. Tg(myl7:GFP) embryo treated with terfenadine. Scale bar 30 μm.

About this article

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DOI

https://doi.org/10.1038/nmeth.3037

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