High-Resolution, Intravital 4D Confocal Time-Lapse Imaging in Avian Embryos Using a Teflon Culture Chamber Design

INTRODUCTIONCell migration is a key aspect of many developmental processes, yet there are relatively few whole-vertebrate embryo culture systems that allow for intravital, high-resolution optical imaging of cell movements, cell-cell interactions, and cell-matrix interactions. Here, we present a protocol for 4D (3D + time), high-resolution confocal imaging of fluorescently labeled cells within living avian embryos. We discuss the culture chamber assembly and interface with a commercially available microscope-stage culture-dish heating system. To demonstrate how the system works, we describe the protocol in use with chick embryos while following individual fluorescently labeled neural crest cells, a major migratory cell population that sorts into complex patterns of discrete streams. By combining the embryo culture directly on glass with confocal 4D time-lapse imaging, we demonstrate that individual neural crest cell migratory behaviors and cell-cell interactions may be visualized for short periods of time (<6 h). This technique can be adapted to study other migratory cell populations or developmental events in whole avian embryos.