This protocol describes the preparation of Caenorhabditis elegans embryos for four-dimensional (4D) imaging. Embryos are excised from gravid hermaphrodites and allowed to adhere directly to a poly-L-lysine-coated coverslip. The coverslip is then used to create a slide chamber in which the embryos are suspended in buffer. These suspended mounts are better for 4D recordings than agar mounts because, although agar mounts produce more uniformly oriented embryos, evaporation from the agar pads tends to shift the embryos gradually over time. 4D spatial registration with suspended mounts is quite stable, and it is possible to digitally reorient embryos within the recorded volume of the three-dimensional (3D) stack during post-processing of the data.
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