Differential performance of aqueous- and ethylic-Lugol's iodine stain to visualize anatomy in μCT-scanned vertebrates.

Museum specimens are an increasingly important tool for studying global biodiversity. With the advent of diffusible iodine-based contrast-enhanced computed tomography (diceCT), researchers can now visualize an organism's internal soft tissue anatomy without the need for physical dissection or other highly destructive sampling methods. However, there are many considerations when deciding which method of staining to use for diceCT to produce the best gray-scale contrast for facilitating downstream anatomical analyses. The general lack of direct comparisons among staining methodologies can make it difficult for researchers to determine which approaches are most appropriate for their study. Here, we compare the performance of ethylic-Lugol's iodine solution with aqueous-Lugol's staining solution across several vertebrate orders to assess differential imaging outcomes. We found that ethylic-Lugol's is better for visualizing muscle attachment to bone but provides overall lower contrast between soft tissue types. Comparatively, aqueous-based Lugol's provides high-contrast imaging among soft tissue types, although bone is more difficult to discern. We conclude that the choice of staining methodology largely depends on the type of anatomical data the researcher wishes to collect, and we provide a decision-based framework for assessing which staining methodology (ethylic or aqueous) is most appropriate for desired imaging results.