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A practical guide to adaptive light-sheet microscopy

Nature Protocols volume 13, pages 2462–2500 (2018)Cite this article

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Abstract

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.

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Acknowledgements

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.

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Author notes

  1. These authors contributed equally: Loïc A. Royer, William C. Lemon, and Raghav K. Chhetri

Authors and Affiliations

  1. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Loïc A. Royer

  2. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA

    William C. Lemon, Raghav K. Chhetri & Philipp J. Keller

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Contributions

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

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

2. Amat, F. et. al. Nat. Methods 11, 951–958 (2014): https://doi.org/10.1038/nmeth.3036

3. Stegmaier, J. et al. Dev. Cell 36, 225–240 (2016): https://doi.org/10.1016/j.devcel.2015.12.028

4. Grimm, J. B. et al. Nat. Methods 14, 987–994 (2017): https://doi.org/10.1038/nmeth.4403

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). https://doi.org/10.1038/s41596-018-0043-4

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