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Multicolor two-photon light-sheet microscopy

Nature Methods volume 11, pages 600–601 (2014)Cite this article

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To the Editor:

Two-photon microscopy is the most effective approach for deep-tissue fluorescence cellular imaging; however, its application to high-throughput or high-content imaging is often hampered by low pixel rates, challenging multicolor excitation and potential cumulative photodamage. To overcome these limitations, we extended our prior work and combined two-photon scanned light-sheet illumination (or two-photon selective-plane illumination microscopy, 2P-SPIM)1 with mixed-wavelength excitation2 to achieve fast multicolor two-photon imaging with negligible photobleaching compared to conventional two-photon laser point-scanning microscopy (2P-LSM). We report on the implementation of this strategy and, to illustrate its potential, recorded sustained four-dimensional (4D: three dimensions + time) multicolor two-photon movies of the beating heart in zebrafish embryos at 28-MHz pixel rates.

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Figure 1: Fast trichromatic two-photon imaging of live embryos using mixed-wavelength light-sheet excitation.

References

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Acknowledgements

We thank A. McMahon and A. Stathopoulos (Caltech) for the mcd8-GFP/H2A-RFP Drosophila line, J.M. Sintes and S. Roth for technical assistance, and T.V. Truong for discussions. We thank M. Liebling and the University of California at Santa Barbara for providing Cardiac Image-Sequence Synchronization Software. This work was supported by the French Agence Nationale de la Recherche (ANR-2010-JCJC-1510-01, ANR-09-PIRI-0013, ANR-11-EQPX-0029), European Union Seventh Framework Programme (Marie Curie International Reintegration Grant no. 268379), Fondation Louis D de l'Institut de France, Human Frontier Science Program and INSERM.

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Authors and Affiliations

  1. Laboratory for Optics and Biosciences, École Polytechnique, Centre National de la Recherche Scientifique (UMR 7645) and Institut National de la Santé et de la Recherche Médicale (U696), Palaiseau, France

    Pierre Mahou, Emmanuel Beaurepaire & Willy Supatto

  2. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique (UMR 1704), Institut National de la Santé et de la Recherche Médicale (U964) and Université de Strasbourg, Illkirch, France

    Julien Vermot

Authors
  1. Pierre Mahou
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  2. Julien Vermot
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  3. Emmanuel Beaurepaire
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  4. Willy Supatto
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Corresponding authors

Correspondence to Emmanuel Beaurepaire or Willy Supatto.

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Competing interests

Work mentioned in this article is the subject of a patent application filed by the Centre National de la Recherche Scientifique, Paris, France, and the Ecole Polytechnique, Palaiseau, France, from P.M., E.B. and W.S. (WO/2013/113861).

Supplementary information

Supplementary Text, Figures and Methods

Supplementary Figures 1–5, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 1501 kb)

Supplementary Video 1

Live 4D and multicolour 2P-SPIM imaging of the beating zebrafish heart (85 fps) 1.2mb (MOV 1212 kb)

Supplementary Video 2

3D cell segmentation and tracking within the entire myocardium of a beating zebrafish heart using multicolour 2P-SPIM imaging data (MOV 6912 kb)

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Mahou, P., Vermot, J., Beaurepaire, E. et al. Multicolor two-photon light-sheet microscopy. Nat Methods 11, 600–601 (2014). https://doi.org/10.1038/nmeth.2963

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