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Diffusible iodine-based contrast-enhanced computed tomography (diceCT) techniques allow visualization of soft tissues of fluid-preserved specimens in three dimensions without dissection or histology. Two popular diceCT stains, iodine-potassium iodide (I 2 KI) dissolved in water and elemental iodine (I 2 ) dissolved in 100% ethanol (EtOH), yield striking results. Despite the widespread use of these stains in clinical and biological fields, the molecular mechanisms that result in color change and radiopacity attributed to iodine staining are poorly understood. Requests to apply these stains to anatomical specimens preserved in natural history museums are increasing, yet curators have little information about the potential for degradation of treated specimens. To assess the molecular effects of iodine staining on typical museum specimens, we compared the two popular stains and two relatively unexplored stains (I 2 KI in 70% EtOH, I 2  in 70% EtOH). House sparrows ( Passer domesticus ) were collected and preserved under uniform conditions following standard museum protocols, and each was then subjected to one of the stains. Results show that the three ethanol-based stains worked equally well (producing fully stained, life-like, publication quality scans) but in different timeframes (five, six, or eight weeks). The specimen in I 2 KI in water became degraded in physical condition, including developing flexible, demineralized bones. The ethanol-based methods also resulted in some demineralization but less than the water-based stain. The pH of the water-based stain was notably acidic compared to the water used as solvent in the stain. Our molecular analyses indicate that whereas none of the stains resulted in unacceptable levels of protein degradation, the bones of a specimen stained with I 2 KI in water demineralized throughout the staining process. We conclude that staining with I 2 KI or elemental I 2  in 70% EtOH can yield high-quality soft-tissue visualization in a timeframe that is similar to that of better-known iodine-based stains, with lower risk of negative impacts on specimen condition.

Chemical effects of diceCT staining protocols on fluid-preserved avian specimens