X-ray microtomographic imaging of intact vertebrate embryos

X-ray microtomography (microCT) is a highly versatile imaging method, and with appropriate contrast stains and sample mounting, it can produce high-quality images of embryos at a wide range of developmental stages. The techniques presented here provide simple and robust methods for gel embedding and microCT scanning of vertebrate embryos. The specimens are fixed by any common method, contrast-stained with phosphotungstic acid (PTA, a common reagent in histological staining), and mounted in agarose gel for scanning. The gel mounting immobilizes the sample effectively during a long scan, and holds the embryo in its liquid medium (70% ethanol), allowing nondestructive three-dimensional (3D) imaging to be followed by, for example, histological processing. An alternative procedure is given for imaging resin-embedded samples intended for transmission electron microscopy (TEM) or subsequent sectioning and ultrastructural analysis. These procedures are expected to lend themselves well to quantitative analyses of mutant and experimental phenotypes and to generating accurate 3D anatomical data for embryological atlases.