title: Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools.
abstract: This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis.
authors: Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, Okazaki Aichi, Japan.
authors: Takehiko
authors: T
authors: Ichikawa
authors: Theoretical Biology Laboratory, RIKEN Advanced Science Institute, Wako-city, Japan.
authors: Kenichi
authors: K
authors: Nakazato
authors: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors: Philipp J
authors: PJ
authors: Keller
authors: Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, Okazaki Aichi, Japan.
authors: Hiroko
authors: H
authors: Kajiura-Kobayashi
authors: Buchmann Institute for Molecular Life Sciences, Goethe Universität Frankfurt, Frankfurt am Main, Germany.
authors: Ernst H K
authors: EH
authors: Stelzer
authors: Theoretical Biology Laboratory, RIKEN Advanced Science Institute, Wako-city, Japan.
authors: Atsushi
authors: A
authors: Mochizuki
authors: Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, Okazaki Aichi, Japan.
authors: Shigenori
authors: S
authors: Nonaka
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian / Q000196:embryology*; D054262:Gastrulation / Q000502:physiology*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007103:Immobilization / Q000379:methods; D051379:Mice; D008853:Microscopy / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*