In mammalian development, the first cell lineages are established in the blastocyst, in which the inner cell mass (ICM) and the trophectoderm (TE) occupy distinct compartment along the embryonic inner-outer axis. The inner and outer cell layers in the embryo are generated during the transition from the 8-cell to 32-cell stage in principle by two dynamic processes, cell division and cell sorting. However, the mechanism by which the spindle orientation and cellular movement are regulated in the early mouse embryo largely remains elusive. This project aims at understanding the mechanical basis of the cell lineage segregation by high-resolution fluorescent live imaging and digital reconstruction of the blastocyst patterning. To minimize photo-damage and bleaching in the embryo, we will adapt selective plane illumination microscopy (SPIM) to the mouse embryo live-imaging. Specifically, we will optimize the optical design and embryo mounting and culture for live-imaging and 4D digital reconstruction. Transgenic fluorescent reporter mice visualizing the cell membrane, the nuclei, cytoskeletons, cell polarity markers and cell adhesion proteins will allow high-resolution tracking of the lineage segregation and understanding of its mechanical basis. Our image processing will extract molecular and cellular dynamics that correlate with cell division or cell sorting along the inner-outer axis, and will establish the underlying mechanistic model. The mechanistic model will allow predicting the impact of cellular physical constraints on cell division plane and movement, and will be evaluated by experimentally testing the prediction by means of micro-manipulation. This study will thus elucidate the mechanical aspect of the ICM and TE segregation for the first time. The developed imaging system and analysis will offer a wider contribution to understanding how cellular mechanical dynamics influence molecular dynamics during development.