Cryo-imaging, as developed by BioInVision, Inc., and fluorescent reporter gene transgenic mice will be used to determine even subtle or rare developmental abnormalities resulting from toxic exposures. By alternately sec- tioning and imaging, the cryo-imaging system will acquire 3D, high-resolution anatomical color and cellular fluorescence images. The cryo-imaging system consists of a large stage, motorized cryostat with special fea- tures for imaging;a modified, bright-field/fluorescence microscope;and a robotic xyz imaging system posi- tioner, all of which are fully automated by a control system. To achieve high throughput, we will modify the sys- tem to acquire images from arrays of as many as 8-50 embryos at a time. There are a very large number of existing fluorescent-reporter-gene transgenic mice which highlight various tissues (smooth muscle cells, endo- thelial cells, neurons, etc.) and emerging transgenic models which fluorescently report upregulation of gene response to toxic stress. With cryo-imaging, we will obtain micron-scale resolution, color and fluorescent vol- umes which will show subtle changes in these engineered mice as a result of toxic exposure. We will develop 3D image visualization/analysis tools allowing us to probe virtual embryos for subtle changes in development. In Phase I, we will demonstrate feasibility using embryos which abundantly expresses EGFP in smooth muscle delineating the fetal vascular, respiratory, and GI systems. Following maternal exposure to TCDD, which is known to affect vascular development, we will analyze fetuses both by conventional manual dissection and by cryo-imaging, and determine the ability of cryo-imaging to detect both gross and subtle variations. Because data is digital, there will be opportunities for computational comparisons of embryos. If this general approach is successful, we will use batteries of fluorescent reporter mice and cryo-imaging to perform toxicology studies providing improved sensitivity and mechanistic information.
We will utilize a new imaging technology and genetically engineered mice to create a very sensitive method for assessing effects of toxins on developing embryos. This research will help drug companies and environmental scientists screen for subtle, rare toxicity effects and to determine general mechanisms of action.
