PUBLICATION
A practical guide to adaptive light-sheet microscopy
- Authors
- Royer, L.A., Lemon, W.C., Chhetri, R.K., Keller, P.J.
- ID
- ZDB-PUB-181028-5
- Date
- 2018
- Source
- Nature Protocols 13(11): 2462-2500 (Other)
- Registered Authors
- Keller, Philipp, Lemon, William, Royer, Loic
- Keywords
- none
- MeSH Terms
-
- Algorithms*
- Animals
- Animals, Genetically Modified
- Drosophila melanogaster/anatomy & histology
- Drosophila melanogaster/cytology
- Embryo, Nonmammalian/cytology
- Embryo, Nonmammalian/ultrastructure*
- Equipment Design/instrumentation
- Guidelines as Topic
- Microscopy, Fluorescence/instrumentation
- Microscopy, Fluorescence/methods*
- Software
- Zebrafish/anatomy & histology
- PubMed
- 30367170 Full text @ Nat. Protoc.
Citation
Royer, L.A., Lemon, W.C., Chhetri, R.K., Keller, P.J. (2018) A practical guide to adaptive light-sheet microscopy. Nature Protocols. 13(11):2462-2500.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping