MATERIALS AND METHODS

One specimen of Sarcoglanis simplex (ANSP 179212) was cleared and double-stained with Alizarin Red S and Alcian Blue (Figure 1.3) prior to MicroCT-scanning. This specimen was then wedged in 2% agarose gel and scanned at low voltage (40kV) and high amperage (250A). The agarose gel prevented dehydration, movement, and shrinkage of the specimen during scanning.

An agar wedge was created to stabilize a specimen using the following protocol: A stock solution of 2% agarose gel was heated in the microwave until it was a clear homogeneous liquid. Agar was then cooled until it was slightly viscous but still clear. Placing the agar into a freezer accelerated cooling. If the agar solution became cloudy, it could not be poured because it tended to clump together. Once slightly viscous, agar was built up around the walls of a small plastic tube in one of two ways, 1) a small amount of liquid agar was poured in a plastic tube that was propped on one side and cooled completely. This step was repeated one or more times creating small shelves against which a specimen could be rested, not allowing it to touch the walls of the tube, or 2) a tube was filled with agar and allowed to cool completely, then a smaller chamber was cut out with a straw. Once an agar chamber was prepared, the specimen was placed into it and slowly covered by warm agar and cooled completely before scanning.

Special care must be taken when scanning 'wet' specimens. It is critical to remove specimens from agar and place them into 70% EtOH (or other curatorial storage medium) within a few days of scanning. The agar remains slippery and relatively brittle when sealed in most mounting tubes for 3-4 days. The agar can be split apart easily allowing the specimen to slide out. If multiple scans are conducted on a single mount, the ends of the tube housing the specimen need to be capped. Capping is typically not enough to prevent dehydration if several days pass between each scan. Agar dehydration can cause a specimen to shift and eventually will make it difficult to remove. In the event that a specimen remains wedged too long, ethanol helps to dissolve the agar. Soaking time in ethanol will vary, but a minimum of one week is suggested. Water and glycerin will not break down the agar.

All scans were conducted on the Skyscan 1172 Microfocus X-radiographic Scanner at Amherst College. Details of scanning parameters are provided in Table 1. MicroCT creates a series of individual X-ray projection images that together form a 360º profile through a specimen. Greyscale values in these images correspond to X-ray attenuation, which varies as a function of variations in density and average atomic number. Greyscale values were determined on a per-specimen basis. Projection images were converted using filtered back-projection into a volume consisting of a stack of X-ray attenuation cross sections, or slices, using the reconstruction software, Cone-Beam Reconstruction v. 2.23 (SKYSCAN, Kontich, Belgium). Dark regions visible in each cross section represented areas of a specimen (i.e., skeletal elements) that are more ossified, and lighter regions typically correspond to soft tissues such as muscle, ligament, or organs, or to voids within samples. Each slice was inverted in color for analysis with the 3D imaging software VGStudio MAX v.1.2.1 (Volume Graphics, Heidelberg, Germany) at The University of Texas at Austin and the Academy of Natural Sciences, Philadelphia. Using thresholding, a range of greyscale values observed on individual bones were identified and digitally isolated from cross-sectional slices. Three-dimensional volume models of structures within the slice volume were produced and volume rendered. In regions where bones are poorly ossified or thin, there are gaps in volume renderings. Some anatomical observations noted in the text will, therefore, not match one-to-one with the figures. Descriptions of osteological features are based on the single C&S specimen and scan data of this specimen. The terminology proposed by de Pinna (1989) is followed. Animations of 3D models were produced using VGStudio MAX and Quicktime Pro.