title: Dual-view plane illumination microscopy for rapid and spatially isotropic imaging.
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.
authors.affiliation: 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.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Yicong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Christensen
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Chandris
authors.affiliation: Biomedical Imaging Research Services Section, Center for Information Technology, NIH, Bethesda, Maryland, USA.
authors.forename: William
authors.initials: W
authors.lastname: Gandler
authors.affiliation: Biomedical Imaging Research Services Section, Center for Information Technology, NIH, Bethesda, Maryland, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: McCreedy
authors.affiliation: Biomedical Imaging Research Services Section, Center for Information Technology, NIH, Bethesda, Maryland, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Bokinsky
authors.affiliation: Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Colón-Ramos
authors.affiliation: Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA.
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Bao
authors.affiliation: Biomedical Imaging Research Services Section, Center for Information Technology, NIH, Bethesda, Maryland, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: McAuliffe
authors.affiliation: Applied Scientific Instrumentation, Eugene, Oregon, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Rondeau
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000196:embryology; D003952:Diagnostic Imaging / Q000295:instrumentation / Q000379:methods; D004625:Embryo, Nonmammalian; D004867:Equipment Design; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D012984:Software; D013997:Time Factors