A practical guide to adaptive light-sheet microscopy. Issue 11 (November 2018)

• Record Type: Journal Article
• Authors: Royer, Loïc; Lemon, William; Chhetri, Raghav; Keller, Philipp
• Is Part Of: Nature protocols. Volume 13:Issue 11(2018)
• Page Start: 2462
• Page End: 2500
• Publication Date: 2018-11
• Subjects: Biology -- Methodology -- Periodicals; Chemistry -- MethodologyPeriodicals; Biology -- Handbooks, manuals, etc; Chemistry -- Handbooks, manuals, etc; 570.28
• DOI: 10.1038/s41596-018-0043-4
• Languages: English
• ISSNs: 1754-2189

Abstract

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. This protocol describes how to implement and apply an adaptive light-sheet microscopy framework (AutoPilot). The procedure can be used to introduce AutoPilot in an existing microscope or to set up a new adaptive multiview light-sheet microscope.