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Nat Protoc. 2014 Nov;9(11):2555-73. doi: 10.1038/nprot.2014.172. Epub 2014 Oct 9.

Dual-view plane illumination microscopy for rapid and spatially isotropic imaging.

Author information

1
1] Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA.
2
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
3
Biomedical Imaging Research Services Section, Center for Information Technology, NIH, Bethesda, Maryland, USA.
4
Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA.
5
Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA.
6
Applied Scientific Instrumentation, Eugene, Oregon, USA.

Abstract

We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.

PMID:
25299154
PMCID:
PMC4386612
DOI:
10.1038/nprot.2014.172
[Indexed for MEDLINE]
Free PMC Article
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