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A practical guide to adaptive light-sheet microscopy


We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope. The protocol describes (i) the mechano-optical implementation of the adaptive imaging hardware, including technical drawings for all custom microscope components; (ii) the algorithms and software library for automated adaptive imaging, including the pseudocode and annotated source code for all software modules; and (iii) the execution of adaptive imaging experiments, as well as the configuration and practical use of the AutoPilot framework. Setup of the adaptive imaging hardware and software takes 1–2 weeks each. Previous experience with light-sheet microscopy and some familiarity with software engineering and building of optical instruments are recommended. Successful implementation of the protocol recovers near diffraction-limited performance in many parts of typical multicellular organisms studied with light-sheet microscopy, such as fruit fly and zebrafish embryos, for which resolution and signal strength are improved two- to fivefold.

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Fig. 1: Overview of the protocol and AutoPilot framework.
Fig. 2: Degrees of freedom of the AutoPilot framework.
Fig. 3: Implementation example of adaptive multiview light-sheet microscopy.
Fig. 4: Example interface for configuring reference planes in a high-speed recording.
Fig. 5: Typical configuration of reference planes in fruit fly and zebrafish embryos.
Fig. 6: Example interface for configuration of AutoPilot parameters.
Fig. 7: Defocus measurements and optimization of resolution with the AutoPilot framework.


  1. 1.

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

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Power, R. M. & Huisken, J. A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373 (2017).

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Stelzer, E. H. Light-sheet fluorescence microscopy for quantitative biology. Nat. Methods 12, 23–26 (2014).

    Article  Google Scholar 

  4. 4.

    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  PubMed  Google Scholar 

  5. 5.

    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  PubMed  Google Scholar 

  6. 6.

    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  PubMed  PubMed Central  Google Scholar 

  7. 7.

    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  PubMed  Google Scholar 

  8. 8.

    Krzic, U., Gunther, S., Saunders, T. E., Streichan, S. J. & Hufnagel, L. Multiview light-sheet microscope for rapid in toto imaging. Nat. Methods 9, 730–733 (2012).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Schmid, B. et al. High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics. Nat. Commun. 4, 2207 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Amat, F. et al. Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data. Nat. Methods 11, 951–958 (2014).

    CAS  Article  PubMed  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  PubMed  Google Scholar 

  12. 12.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Panier, T. et al. Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy. Front. Neural Circuits 7, 65 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

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

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Wolf, S. et al. Whole-brain functional imaging with two-photon light-sheet microscopy. Nat. Methods 12, 379–380 (2015).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

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

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Voie, A. H., Burns, D. H. & Spelman, F. A. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Fuchs, E., Jaffe, J., Long, R. & Azam, F. Thin laser light sheet microscope for microbial oceanography. Opt. Express 10, 145–154 (2002).

    Article  PubMed  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  PubMed  Google Scholar 

  21. 21.

    Royer, L. A. et al. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat. Biotechnol. 34, 1267–1278 (2016).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Wolff, C. et al. Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb. Elife 7, e34410 (2018).

  23. 23.

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

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Khairy, K., Lemon, W., Amat, F. & Keller, P. J. A preferred curvature-based continuum mechanics framework for modeling embryogenesis. Biophys. J. 114, 267–277 (2018).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Stegmaier, J. et al. Real-time three-dimensional cell segmentation in large-scale microscopy data of developing embryos. Dev. Cell 36, 225–240 (2016).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Grimm, J. B. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat. Methods 14, 987–994 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Liu, Z. & Keller, P. J. Emerging imaging and genomic tools for developmental systems biology. Dev. Cell 36, 597–610 (2016).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

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

    Article  PubMed  Google Scholar 

  29. 29.

    Liu, T. L. et al. Observing the cell in its native state: imaging subcellular dynamics in multicellular organisms. Science 360, 6386 (2018).

    Article  Google Scholar 

  30. 30.

    Baumgart, E. & Kubitscheck, U. Scanned light sheet microscopy with confocal slit detection. Opt. Express 20, 21805–21814 (2012).

    Article  PubMed  Google Scholar 

  31. 31.

    Fahrbach, F. O. & Rohrbach, A. Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media. Nat. Commun. 3, 632 (2012).

    Article  PubMed  Google Scholar 

  32. 32.

    de Medeiros, G. et al. Confocal multiview light-sheet microscopy. Nat. Commun. 6, 8881 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Silvestri, L., Bria, A., Sacconi, L., Iannello, G. & Pavone, F. S. Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain. Opt. Express 20, 20582–20598 (2012).

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Rohrbach, A. Artifacts resulting from imaging in scattering media: a theoretical prediction. Opt. Lett. 34, 3041–3043 (2009).

    Article  PubMed  Google Scholar 

  35. 35.

    Huisken, J. & Stainier, D. Y. Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt. Lett. 32, 2608–2610 (2007).

    Article  PubMed  Google Scholar 

  36. 36.

    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  PubMed  Google Scholar 

  37. 37.

    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  PubMed  PubMed Central  Google Scholar 

  38. 38.

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

    CAS  Article  Google Scholar 

  39. 39.

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

    Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Dunsby, C. Optically sectioned imaging by oblique plane microscopy. Opt. Express 16, 20306–20316 (2008).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Bouchard, M. B. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photonics 9, 113–119 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    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  PubMed  Google Scholar 

  43. 43.

    Keller, P. J., Pampaloni, F. & Stelzer, E. H. Three-dimensional preparation and imaging reveal intrinsic microtubule properties. Nat. Methods 4, 843–846 (2007).

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Preibisch, S., Saalfeld, S., Schindelin, J. & Tomancak, P. Software for bead-based registration of selective plane illumination microscopy data. Nat. Methods 7, 418–419 (2010).

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Preibisch, S. et al. Efficient Bayesian-based multiview deconvolution. Nat. Methods 11, 645–648 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Balazs, B., Deschamps, J., Albert, M., Ries, J. & Hufnagel, L. A real-time compression library for microscopy images. Preprint at bioRxiv, (2017).

  47. 47.

    Freeman, J. et al. Mapping brain activity at scale with cluster computing. Nat. Methods 11, 941–950 (2014).

    CAS  Article  PubMed  Google Scholar 

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We thank all members of the Keller Lab for extensive testing of the AutoPilot framework and for their contributions to the development of this method, M. Coleman (Coleman Technologies) for custom microscope operating software, B. Coop and the jET team at the Janelia Research Campus for mechanical designs and custom mechanical parts, and M. Staley for help with producing the video demonstrating the specimen-embedding procedure. This work was supported by the Howard Hughes Medical Institute and the Chan Zuckerberg Biohub.

Author information




All authors contributed to the development of the protocol and the writing of the manuscript.

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 provisional US patent application 62,354,384 for adaptive light-sheet microscopy on 24 June 2016. P.J.K. holds US patent 9,404,869 for simultaneous multiview light-sheet microscopy, issued 2 August 2016.

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Key references using this protocol

1. Royer, L. A. et al. Nat. Biotechnol. 34, 1267–1278 (2016):

2. Amat, F. et. al. Nat. Methods 11, 951–958 (2014):

3. Stegmaier, J. et al. Dev. Cell 36, 225–240 (2016):

4. Grimm, J. B. et al. Nat. Methods 14, 987–994 (2017):

Supplementary information

Supplementary Data 1

Supplementary Data 2

Supplementary Video 1

Embedding of a specimen for imaging in the light-sheet microscope

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Royer, L.A., Lemon, W.C., Chhetri, R.K. et al. A practical guide to adaptive light-sheet microscopy. Nat Protoc 13, 2462–2500 (2018).

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