Enhancing CT imaging: A safe protocol to stain and de-stain rare fetal museum specimens using diffusible iodine-based staining (diceCT).

Computed tomography (CT) scanning is being increasingly employed in the study of natural history, particularly to investigate the internal anatomy of unique specimens in museum collections. Different techniques to enhance the contrast between tissues have been developed to improve the quality of the scans while preserving the integrity of these rare specimens. Diffusible iodine-based contrast enhanced computed tomography (diceCT) was found to be particularly effective and reversible for staining tissues in formalin preserved specimens. While it can also be effectively employed to stain ethanol-preserved specimens of small size, the reversibility of this process and the applicability to large-bodied animals has never been thoroughly tested. Here, we describe a novel diceCT protocol developed to stain and de-stain ethanol-preserved prenatal specimens of baleen whales (Mysticeti, Cetacea). These large (10-90 cm in length only considering early fetal stages) specimens present unique challenges as they are rare in collections and irreplaceable, therefore it is imperative to not damage them with the staining process. Before trying this protocol on baleen whales' specimens, we conducted a pilot study on commercially available fetal pigs using the same parameters. This allowed us to optimize the staining time to obtain the best results in CT scanning and to test first-hand the effect of de-staining on ethanol-based specimens. External coloration of the specimens is also a concern with iodine-staining, as stained specimens assume a bright red color that needs to be removed from both internal and external tissues before they can be stored. To test the reversibility of the stain in ethanol-preserved specimens with fur, we also conducted a small experiment using commercially acquired domestic mice. After these trials were successful, we applied the staining and de-staining protocol to multiple fetal specimens of mysticetes up to 90 cm in length, both ethanol- and formalin-preserved. Specimens were soaked in a solution of 1% pure iodine in 70% ethanol for 1-28 days, according to their size. After scanning, specimens are soaked in a solution of 3% sodium thiosulfate in 70% ethanol that is able to completely wash out the iodine from the tissues in a shorter time frame, between a few hours and 14 days. The same concentrations were used for formalin-preserved specimens, but DI water was used as solvent instead of ethanol. The staining technique proved particularly useful to enhance the contrast difference between cartilage, mineralized bone, teeth, and the surrounding soft tissues even when the specimens where scanned in medical-grade CT scans. The specimens did not suffer any visible damage or shrinkage due to the procedure, and in both the fetal samples and in the mice the color of the stain was completely removed by the de-staining process. We conclude therefore that this protocol can be safely applied to a variety of ethanol-preserved museum specimens to enhance the quality of the CT scanning and highlight internal morphological features without recurring to dissection or other irreversible procedures. We also provide tips to best apply this protocol, from how to mix the solutions to how to minimize the staining time.