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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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.
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
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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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