Time-lapse imaging of live Phallusia embryos for creating 3D digital replicas

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article provides a method to fluorescently label and image structures of interest that will subsequently be reconstructed, such as cell membranes or nuclei. The protocol describes live imaging of Phallusia mammillata embryos, which are robust, colorless, and optically transparent with negligible autofluorescence. Their diameter ranges from 100 µm to 120 µm, which allows time-lapse microscopy of whole embryos using two-photon microscopy with a high-resolution objective. Although two-photon imaging is described in detail, any imaging technology that results in a z-stack may be used. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes.