The authors declare that they have no competing interests.
ZL carried out the staining experiment, led design of the study, and drafted the manuscript. JAC and RAK participated in the design of the study, analysis of data, and drafted the manuscript. MWC participated in the acquisition and analysis of the CT data. FY participated in the modeling design and simulation analyses. All authors contributed to the writing of the manuscript.
Iodine-based solutions have long been known to be effective in aiding the differentiation among soft tissues in both fundamental anatomical research and for clinical diagnoses. Recently the combination of this particular contrasting agent with micro-computed tomography (micro-CT) has resulted in an array of high-quality image data, in which anatomical structures not visible in conventional CT can be identified and quantified. However, there has been only limited data available to inform detailed protocols for staining large specimens. Further, modeling of the staining mechanism has focused on simple diffusion processes.
A low concentration of iodine-based buffered formalin solution with a long staining period was used to visualize soft-tissue structures in a large goose head. The staining effect was analyzed by serially measuring the micro-CT profiles across coronal sections throughout the staining period. Regular replacement of the staining solution combined with a longer staining period significantly improved contrast within tissues. A simplified one-dimensional Diffusion-Sorption model with a three-zone domain was used to simulate the diffusion process by calculating the concentration profile of iodine across the adductor region, which fits well with the experiment data. Observations of changes in the concentration of the staining agent and simulation results suggest that the sorption of iodine by tissues significantly affects the effective diffusion coefficient for the contrasting agent.
The Diffusion-Sorption model better explains previously reported difficulties in staining large samples comprised of tissues with high partition coefficients (K d ). Differences in partition coefficient (K d ), bulk density (ρ b ), and porosity (θ) could further explain the observed variation in staining rate and maximal staining effect among different tissues. Recommended protocols for staining large specimens are detailed.