Three‐dimensional visualization of cranial soft tissues in embryonic and early postnatal mice using phosphotungstic acid enhanced microCT

Abstract

Acquiring three‐dimensional imaging of animal specimens utilized in genetic, developmental, and evolutionary studies has typically been based on microCT (bone and mineralized tissues) and magnetic resonance microscopy (soft and un‐mineralized tissues). However, recent advances in microCT imaging have allowed for 1–5 μm resolution for small biological specimens, while typical microMRI imaging can generally only produce data with a resolution of approximately 30–40 μm. In order to achieve high‐resolution 3D visualization of soft tissues, the use of various contrast agents have been proposed, including iodine, osmium, and phosphotungstic acid (PTA). We investigated protocols for using PTA to stain soft tissues of the head in embryonic and early postnatal mice to enhance visualization of soft tissues by microCT imaging. Protocols for mice between embryonic day 13.5 and postnatal day 7 have been developed that allow for the visualization, segmentation, quantification, and analysis of soft tissue structures. Stained specimens were mounted in a 50:50 mix of polyester and paraffin waxes within a small tube and scanned on the GE v|tome|x L300 scanner in the Center for Quantitative Imaging at Pennsylvania State University with voxel dimensions between 1 and 10 μm, depending on specimen size. Multiple soft tissue structures, including the eye, inner ear, cartilage, muscle, glandular tissue, and brain, are easily differentiated in the resultant scans. Images of PTA‐stained specimens can be superimposed with microCT images of the same specimen before PTA staining to enable the study of relationships between hard and soft tissues in the same individual. This approach to both 2D and 3D visualization of soft tissues in murine models has immediate implications for understanding developmental processes and integration between hard and soft tissues of the cranium.Support or Funding InformationNational Science Foundation Grant BCS‐1731909, and NIDCR R01DE027677 and R01 DE022988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.