MicroCT Imaging and 3D Reconstruction Protocol for Hard and Soft tissue.
Embryos investigated include wildtype and various conditional knock out models. Samples are harvested on days E13.5 - E18.5 post coitus or at P0.
Samples are washed in 1x PBS and rocked at RT for 30 minutes to 1 hour. All samples are denuded and fixed in 10% NBF or 4% PFA at room temperature (RT) for at least 5 days prior to submission to the University of Southern California Molecular Imaging Center. MicroCT analysis is performed using a SCANCO μCT50 (Scanco V1.28). The microCT images are acquired from embryos with the x-ray source at 70 kVp, 250 μA (bone) and 114uA (Soft), with rotation time of 1250ms (soft) and 300ms (hard). Typical spatial resolution was 10-16 μm per voxel 10μm voxel size in one bed position.
Hard tissue/Bone microCT scans: Dataset of the skull were acquired prior to staining of the soft tissues. Scans obtained at 20μm per slice thickness, resolution at 50 pixels per pixel, and 16 bits per pixel.
Soft tissue staining protocol: Samples are placed in Phosphotungstic acid (PTA)/PBS solution in a volume that is at least 8x the specimen size. Rock the sample at RT for 3 days. Place the sample in PTA solution in a volume that is at least 8x the specimen size. Rock the sample at RT for 3 days. Place the sample in 1x PBS and rock for 30 minutes at RT.
Soft tissue microCT Scan optimization: Perform QC scan to ensure the entire sample is stained. QC scans are obtained at 30 microns. If sample is stained, proceed to high resolution scan. High resolution scans are 10 microns or less. If the sample is under/over stained, place back into PBS and rock at room temperature. Monitor stain everyday or every other day by performing QC scans.
Imaging Analysis and processing: CT scans were reconstructed using Cobra 6.9.4 (Exxim Computing Corporation, Pleasanton, CA) into CT DICOM datasets. Nifti files are reconstructed and edited in 3D using AVIZO 7.1 (Visualization Sciences Group). (http://www.vsg3d.com/avizo/standard). Avizo software runs on systems with a minimum of 4GB RAM and 500GB hard drive and ATI Radeon HD 5450 graphics card in conjunction with a Wacom Cintiq 22HD tablet for image tracing. Defined craniofacial anatomy files and images are manually segmented by selecting specific anatomical structures and assigning cuboidal “voxels” to a label. All files are submitted to the FaceBase Hub via a shared dropbox folder or secure FTP site.