Comparison and Evaluation of the Effectiveness of Two Approaches of Diffusible Iodine-Based Contrast-Enhanced Computed Tomography (diceCT) for Avian Cephalic Material.

Diffusible iodine-based contrast-enhanced computed tomography presents a comparatively new tool kit for imaging fine-scale three-dimensional phenotypes that is rapidly becoming standard anatomical practice. However, relatively few studies have attempted to look at subtle differences in staining protocols or attempted to model tissue reactions to gain insight into staining mechanisms. Here, two iodine-based contrast agents, iodine-ethanol (I2 E) and iodine-potassium iodide (I2 KI) in neutral buffered formalin , were applied to avian cephalic specimens to investigate their effectiveness. We found that the two solutions had markedly different results for staining of mineralized skeletal tissues (i.e., bone). Other tissues, including muscles, epithelia, and common connective tissues (e.g., lamina propria) were assessed individually and show minor differences in the sorption of iodine. Numerical simulations suggest that different results from I2 E and I2 KI-formaldehyde staining are due to different partition coefficients and retardation factors of tissues, fixation effects, as well as distinct iodine diffusion and sorption patterns. We found a clear positive relationship between glycogen concentration and grayscale values measured within muscle, epithelia, nervous tissues, and glands. We also found the use of ethanol for tissue fixation and following I2 E staining outperforms I2 KI-formaldehyde by providing higher efficiency for acquiring greater contrast both between different soft tissues and between mineralized and nonmineralized tissues.