Jumping on the 3D bandwagon that has infested Hollywood, I wanted to introduce the Anole Annals community to the newest tool being employed to study Anole diversity and evolution, High Resolution X-ray Computed Tomography, or CT scanning for short.
HRXCT is a tool that uses x-rays to visualize the internal geometries of opaque objects. It is similar to the CAT scan you would get at a hospital, but with high-power x-rays so higher resolution. It is perfect for museum specimens because it is non-destructive; you can study skeletal morphology without removing skin or flesh, unlike the skeletonizing or clearing and staining methods as previously described here. In this first blog post on HRXCT of anoles, I shall explain how the scan process works and how the data are collected.
First, the specimens are prepared for scanning. I have developed an approach whereby I scan several lizards of the same size in one go. This maximises my scanning productivity, since in a 1 hour scan, I get 6 lizard skeletons. Scan resolution is measured in voxels (volume pixels), and is determined by the size of the object in the 2000 x 2000 pixel detector panel. The resolution is the same for 1 lizard as it is for 6, as long as they are all of similar size. for 45mm snout-vent length lizards, I get about 0.045mm resolution. Below is an overview of the sample mounting; the alcohol-preserved anoles are wrapped in alcohol-soaked cheese cloth, sealed in plastic bags, and mounted in oasis foam.
Inside the scanner, 3142 x-ray projections are taken as the specimen rotates 360 degrees. The computer receiving the projections then starts the reconstruction; projections are radially aligned and reconstructed into a volume.
Then using software for editing CT volumes, I segment each lizard from the volume and save it as an image stack. On the slices above, you will see different the different materials are represented by different gray values – black is air, grey is soft tissue, white is bone. I segment the skeleton by thresholding the slices and keeping only the “white” voxels. This results in a rendered skeleton.
These 3D skeletons are very detailed and reveal the complex geometry of skeletal variation in Anolis. Watch this space for some of the cool things we find using CT, and how the CT scans will aid our research on anoles. And bonus points to anyone you can name the species pictured here!