Live Imaging of Mouse Embryonic Stem Cells and Embryos: Protocols for In Vitro Culture, Microscopy, and Image Data...

Live imaging is a cornerstone technique in developmental and stem cell biology, enabling visualization of dynamic features in cells, tissues, and embryos. Here, we present current protocols for live imaging of mouse pluripotent stem cells (mESCs) and pre- and postimplantation embryos using confocal and light-sheet microscopy. These methods integrate optimized ex utero culture conditions, mounting geometries, and environmental control systems to support long-term, physiologically relevant imaging with minimal phototoxicity. We outline approaches for imaging pluripotent stem cells under distinct culture states, as well as strategies for embryo imaging compatible with normal growth and morphogenesis. Finally, we discuss computational workflows for image processing, segmentation, and cell tracking that enable quantitative analysis of lineage dynamics. Together, these protocols provide a comprehensive and reproducible framework for investigating cell state transitions and morphogenetic processes in mammalian development.