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J Vis Exp. 2019 Jun 6;(148). doi: 10.3791/59533.

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution.

Author information

1
Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine; WormGUIDES.org.
2
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health; WormGUIDES.org.
3
Developmental Biology Program, Sloan Kettering Institute; WormGUIDES.org.
4
Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center; WormGUIDES.org; wmohler@neuron.uchc.edu.
5
Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine; WormGUIDES.org; Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico; daniel.colon-ramos@yale.edu.

Abstract

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

PMID:
31233035
DOI:
10.3791/59533

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