Article | Published:

Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms

Nature Biotechnology volume 34, pages 12671278 (2016) | Download Citation

Abstract

Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish (Danio rerio) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.

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References

  1. 1.

    & Advances in whole-embryo imaging: a quantitative transition is underway. Nat. Rev. Mol. Cell Biol. 15, 327–339 (2014).

  2. 2.

    Imaging morphogenesis: technological advances and biological insights. Science 340, 1234168 (2013).

  3. 3.

    , & Quantitative analysis of embryogenesis: a perspective for light sheet microscopy. Dev. Cell 23, 1111–1120 (2012).

  4. 4.

    & Faster fluorescence microscopy: advances in high speed biological imaging. Curr. Opin. Chem. Biol. 20, 46–53 (2014).

  5. 5.

    Light sheet fluorescence microscopy: a review. J. Histochem. Cytochem. 59, 129–138 (2011).

  6. 6.

    & Light sheet microscopy for real-time developmental biology. Curr. Opin. Genet. Dev. 21, 566–572 (2011).

  7. 7.

    Light-sheet fluorescence microscopy for quantitative biology. Nat. Methods 12, 23–26 (2015).

  8. 8.

    et al. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013).

  9. 9.

    et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

  10. 10.

    et al. Whole-animal functional and developmental imaging with isotropic spatial resolution. Nat. Methods 12, 1171–1178 (2015).

  11. 11.

    et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 108, 17708–17713 (2011).

  12. 12.

    , , , & Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).

  13. 13.

    , , , & Rapid 3D light-sheet microscopy with a tunable lens. Opt. Express 21, 21010–21026 (2013).

  14. 14.

    , , & Quantitative fluorescence imaging of protein diffusion and interaction in living cells. Nat. Biotechnol. 29, 835–839 (2011).

  15. 15.

    et al. Whole-central nervous system functional imaging in larval Drosophila. Nat. Commun. 6, 7924 (2015).

  16. 16.

    , , & Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat. Methods 9, 755–763 (2012).

  17. 17.

    , , & Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development 139, 3242–3247 (2012).

  18. 18.

    , , & Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

  19. 19.

    et al. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat. Methods 7, 637–642 (2010).

  20. 20.

    , , , & Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

  21. 21.

    et al. Rapid adaptive optical recovery of optimal resolution over large volumes. Nat. Methods 11, 625–628 (2014).

  22. 22.

    et al. Multiplexed aberration measurement for deep tissue imaging in vivo. Nat. Methods 11, 1037–1040 (2014).

  23. 23.

    et al. Calcium imaging of neural circuits with extended depth-of-field light-sheet microscopy. Opt. Lett. 41, 855–858 (2016).

  24. 24.

    et al. SPED light sheet microscopy: fast mapping of biological system structure and function. Cell 163, 1796–1806 (2015).

  25. 25.

    & Image-based calibration of a deformable mirror in wide-field microscopy. Appl. Opt. 49, 2030–2040 (2010).

  26. 26.

    & Aberrations and their correction in light-sheet microscopy: a low-dimensional parametrization. Biomed. Opt. Express 4, 1654–1661 (2013).

  27. 27.

    et al. High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM. Sci. Rep. 5, 16898 (2015).

  28. 28.

    , , , & 3D adaptive optics in a light sheet microscope. Opt. Express 20, 13252–13261 (2012).

  29. 29.

    et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

  30. 30.

    & Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy. Neuron 85, 462–483 (2015).

  31. 31.

    & The smart and gentle microscope. Nat. Biotechnol. 33, 815–818 (2015).

  32. 32.

    , , , & Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat. Methods 8, 757–760 (2011).

  33. 33.

    , , & A simple scanless two-photon fluorescence microscope using selective plane illumination. Opt. Express 18, 8491–8498 (2010).

  34. 34.

    et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

  35. 35.

    , & Microscopy with self-reconstructing beams. Nat. Photonics 4, 780–785 (2010).

  36. 36.

    Microtubule Dynamic Instability Analyzed in Three Dimensions Over Time with Selective Plane Illumination Microscopy and Modeling of Yeast Sporulation. Diploma thesis, University of Heidelberg. (2005).

  37. 37.

    Physical Properties of Tissue: A Comprehensive Reference Book (Academic Press Inc., San Diego, 1990).

  38. 38.

    et al. Efficient processing and analysis of large-scale light-sheet microscopy data. Nat. Protoc. 10, 1679–1696 (2015).

  39. 39.

    , , , & Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol. 92, 14.20.1–14.20.17 (2010).

  40. 40.

    et al. OpenSPIM: an open-access light-sheet microscopy platform. Nat. Methods 10, 598–599 (2013).

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Acknowledgements

We thank all members of the Keller laboratory for extensive testing of the AutoPilot framework; Tzumin Lee (Janelia Research Campus) for deadpanEE–Gal4 Drosophila stocks; Misha Ahrens for Tg(elavl3:GCaMP6f) zebrafish stocks; Nicola Maghelli for help with illustrations; Martin Weigert for 3D rendering software; Vanessa de Oliveira Carlos for D. rerio graphics; and Misha Ahrens, Chen Wang, Katie McDole, Kaspar Podgorski, Na Ji, Eric Betzig and Iain Patten for helpful discussions and thoughtful comments on the manuscript. This work was supported by the Howard Hughes Medical Institute, the Max Planck Institute for Cell Biology and Genetics and the German Federal Ministry of Research and Education (BMBF) under the funding code 031A099.

Author information

Affiliations

  1. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.

    • Loïc A Royer
    • , William C Lemon
    • , Raghav K Chhetri
    • , Yinan Wan
    •  & Philipp J Keller
  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

    • Loïc A Royer
    •  & Eugene W Myers
  3. Coleman Technologies Incorporated, Newtown Square, Pennsylvania, USA.

    • Michael Coleman

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Contributions

P.J.K. and L.A.R. conceived of the research and developed the AutoPilot framework. L.A.R. designed and wrote the AutoPilot core algorithms. M.C. implemented the microscope control software, with input from P.J.K. and L.A.R. R.K.C. implemented the light-sheet microscope with digitally adjustable degrees of freedom. W.C.L. performed adaptive imaging experiments of Drosophila embryogenesis and the zebrafish larval brain. W.C.L. and Y.W. performed adaptive imaging experiments of zebrafish embryogenesis. P.J.K. supervised the project. L.A.R. and P.J.K. wrote the paper, with input from all authors.

Competing interests

P.J.K., R.K.C. and L.A.R. filed a provisional US patent application for adaptive light-sheet microscopy on 24 June 2016 (application number 62,354,384).

Corresponding authors

Correspondence to Loïc A Royer or Philipp J Keller.

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DOI

https://doi.org/10.1038/nbt.3708

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