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A practical guide to adaptive light-sheet microscopy
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DOI：10.1038/s41596-018-0043-4.png)
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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.
Keyword:
PLANE ILLUMINATION MICROSCOPY
FLUORESCENCE MICROSCOPY
DEVELOPING EMBRYOS
BRAIN ACTIVITY
SCALE
CELL
RESOLUTION
RECONSTRUCTION
EXCITATION
ORGANISMS
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Visualizing Whole-Brain Activity and Development at the Single-Cell Level Using Light-Sheet Microscopy.png)
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