Structural Stabilization of Tissue for Embryo Phenotyping Using Micro-CT with Iodine Staining

Abstract

The International Mouse Phenotyping Consortium has been established to conduct large-scale phenotyping of the approximately 23,000 single-gene knockout mice generated by the International Knockout Mouse Consortium to investigate the role of each gene in the mouse genome. Of the generated mouse lines, 30% are predicted to be embryonic lethal, requiring the implementation of imaging techniques and analysis tools specific to late gestation mouse embryo phenotyping. A well-adopted technique combines the use of iodinated contrast solutions and micro-computed tomography imaging. This simple iodine immersion technique provides superior soft-tissue contrast enhancement, however, the hypertonic nature of iodine promotes dehydration causing moderate to severe tissue deformation. Here, we combine the stabilizing properties of a hydrogel mesh with the enhanced contrast properties of iodine. The protocol promotes cross linking of tissue through formaldehyde fixation and the linking of hydrogel monomers to biomolecules. As a result, the hydrogel supports tissue structure and preserves its conformation taking advantage of iodine-enhanced soft tissue contrast to produce high quality mouse embryo images with minimal tissue distortion. Hydrogel stabilization substantially reduces intersample anatomical variation of mature mouse embryos subjected to iodine preparation protocols. A 20% and 50% reduction in intersample variation of normalized brain and lung volume is achieved through hydrogel stabilization, as well as a 20% reduction in variation in overall embryo anatomy as measured through image registration methods. This increases the sensitivity of computer automated analysis to reveal significant anatomical differences between mutant and wild-type mice.