Four-dimensional fluorescent imaging of embryonic quail development

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 (PE) microscopy enables researchers to acquire volumetric images in three dimensions (x, y, and z) plus time (t). Here, we present techniques for acquisition and analysis of three-dimensional time-lapse data. Both confocal microscopy and 2PE microscopy techniques are used. In addition, data processing for tiled image stitching and time-lapse analysis is discussed. Additionally, the development of a new 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. Here we also outline the preparation in vitro of these transgenic quail embryos for imaging.