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

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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Figure 1: Spatiotemporally adaptive light-sheet microscopy.
Figure 2: Spatiotemporally adaptive imaging of Drosophila embryonic development.
Figure 3: Spatiotemporally adaptive imaging of zebrafish embryonic development.
Figure 4: Spatiotemporally adaptive imaging of dynamic gene expression patterns.
Figure 5: Spatiotemporally adaptive optimization of the 3D light-sheet path in vivo.
Figure 6: Spatiotemporally adaptive whole-brain functional imaging in larval zebrafish.

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    Article  Google Scholar 

  3. 3

    Höckendorf, B., Thumberger, T. & Wittbrodt, J. Quantitative analysis of embryogenesis: a perspective for light sheet microscopy. Dev. Cell 23, 1111–1120 (2012).

    Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

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

    Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Wu, Y. 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).

    CAS  Article  Google Scholar 

  12. 12

    Ahrens, M.B., Orger, M.B., Robson, D.N., Li, J.M. & Keller, P.J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).

    CAS  Article  Google Scholar 

  13. 13

    Fahrbach, F.O., Voigt, F.F., Schmid, B., Helmchen, F. & Huisken, J. Rapid 3D light-sheet microscopy with a tunable lens. Opt. Express 21, 21010–21026 (2013).

    Article  Google Scholar 

  14. 14

    Capoulade, J., Wachsmuth, M., Hufnagel, L. & Knop, M. Quantitative fluorescence imaging of protein diffusion and interaction in living cells. Nat. Biotechnol. 29, 835–839 (2011).

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

    Tomer, R., Khairy, K., Amat, F. & Keller, P.J. Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat. Methods 9, 755–763 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Kaufmann, A., Mickoleit, M., Weber, M. & Huisken, J. Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development 139, 3242–3247 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Keller, P.J., Schmidt, A.D., Wittbrodt, J. & Stelzer, E.H.K. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

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

    Article  Google Scholar 

  26. 26

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

    Article  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 28

    Bourgenot, C., Saunter, C.D., Taylor, J.M., Girkin, J.M. & Love, G.D. 3D adaptive optics in a light sheet microscope. Opt. Express 20, 13252–13261 (2012).

    Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

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

    CAS  Article  Google Scholar 

  32. 32

    Truong, T.V., Supatto, W., Koos, D.S., Choi, J.M. & Fraser, S.E. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat. Methods 8, 757–760 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Palero, J., Santos, S.I., Artigas, D. & Loza-Alvarez, P. A simple scanless two-photon fluorescence microscope using selective plane illumination. Opt. Express 18, 8491–8498 (2010).

    CAS  Article  Google Scholar 

  34. 34

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

    CAS  Article  Google Scholar 

  35. 35

    Fahrbach, F.O., Simon, P. & Rohrbach, A. Microscopy with self-reconstructing beams. Nat. Photonics 4, 780–785 (2010).

    CAS  Article  Google Scholar 

  36. 36

    Keller, P.J. 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

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

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

    Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol. 92, 14.20.1–14.20.17 (2010).

    Google Scholar 

  40. 40

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

    CAS  Article  Google Scholar 

Download references

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.

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Authors

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.

Corresponding authors

Correspondence to Loïc A Royer or Philipp J Keller.

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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).

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–19 and Supplementary Tables 1–11 (PDF 16230 kb)

Supplementary Software (ZIP 173448 kb)

Perturbation benchmark of spatiotemporally adaptive imaging performance (AVI 29264 kb)

Spatiotemporally adaptive imaging of Drosophila embryogenesis (AVI 51053 kb)

Recovery of high spatial resolution in Drosophila adaptive imaging (AVI 50902 kb)

Recovery of cellular resolution in deep tissue layers by adaptive imaging (AVI 4794 kb)

Spatiotemporally adaptive imaging of zebrafish embryogenesis (AVI 26395 kb)

Recovery of high spatial resolution in zebrafish adaptive imaging (AVI 7809 kb)

Quantification of resolution improvements in zebrafish adaptive imaging (AVI 7684 kb)

Spatiotemporally adaptive two-color imaging of neural development (AVI 36649 kb)

Spatiotemporally adaptive whole-brain functional imaging in larval zebrafish (AVI 17161 kb)

Example of system drift during non-adaptive long-term imaging (AVI 28293 kb)

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Royer, L., Lemon, W., Chhetri, R. et al. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat Biotechnol 34, 1267–1278 (2016). https://doi.org/10.1038/nbt.3708

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