Traditionally, our understanding of developmental biology has been based on the fixation and study of embryonic samples. Detailed microscopic scrutiny of static specimens at varying ages allowed for anatomical assessment of tissue development. The advent of confocal and two-photon excitation (2PE) microscopy enables researchers to acquire volumetric images in three dimensions (x, y, and z) plus time (t). The development of transgenic Japanese quail has provided an embryonic model system that is more easily accessible than mammalian models and more efficient to breed than the classic avian model, the chicken. This protocol describes the methods necessary to prepare transgenic quail embryos for imaging in vitro, and for acquiring, processing, and analyzing three-dimensional (3D) time-lapse images.