4D 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 (2PE) 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 (3D) time-lapse data. Both confocal microscopy and 2PE microscopy techniques are used. Data processing for tiled image stitching and time-lapse analysis is also discussed. The development of a transgenic Japanese quail system, as discussed here, has provided an embryonic model that is more easily accessible than mammalian models and more efficient to breed than the classic avian model, the chicken.