| Abstract: |
Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as postimplantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal newinsights intomouse embryogenesis. Althoughwe have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants. |
