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

Abstract

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 ethanolbased stains worked equally well (producing fully stained, lifelike , 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.

DiceCT applied to fossilized hard tissues: A preliminary case study using a miocene bird

“…Two explanations are possible for the material studied here: (1) the energy (keV) used during the nano‐CT scanning was not appropriate to detect the fine differences in calcium content between bone and calcified cartilage (i.e., the KeV was either too high or too low); (2) iodine‐ethanol somehow decreased the natural contrasts between the ECM of bone and calcified cartilage. Early et al, 2020 showed that bone demineralization occurs during I 2 E staining in extant specimens, but more data needs to be collected to test whether I 2 E can also demineralize fossil bone and calcified cartilage and whether this is a process that must be taken in consideration when trying to understand changes in contrasts for fossil tissues.…”

“…The last limitation concerns molecular tests. If molecular or chemical works need to be performed on the fossil after immersion into iodine, its contamination should be taken into consideration, as it will most likely interact with originally preserved molecules in a process that may be nonreversible (see Early et al, 2020). It is also possible that ethanol removes or destroys some fossilized biomolecules and that many of them might get lost into the staining solution.…”

“…This previously unknown molecular mechanism of iodine staining (Early et al, 2020) adds greatly to our understanding of iodine interactions with organic molecules, however many questions remain. For example, it is still unclear whether or not iodine binds to collagen I even though it is the most abundant molecule in bone (Early et al, 2020), or whether it can also bind to lipids and/or polysaccharides as suggested earlier .…”

“…The authors found that I 2 E outperformed Lugol's iodine, providing higher and better contrast between different soft tissues but also between mineralized and soft tissues (Li et al, 2016). More recently, it was found that I 2 E in 70% ethanol is also a good contrast enhancer for both bone and soft‐tissues (Early et al, 2020).…”

“…In modern tissues, it was recently discovered that the iodination of amino acids (in avian bone and muscle tissues) is one mechanism that can explain how I 2 E staining works (Early et al, 2020). Here, we did not further investigate this issue at the proteomic level, but we further investigated possible mechanisms of iodine staining in the fossil tissues using electron dispersive spectroscopy (EDS).…”