A guide for optimal iodine staining and high-throughput diceCT scanning
in snakes
Abstract
Diffusible iodine-based contrast-enhanced Computed-Tomography (diceCT)
visualizes soft-tissue from microCT (µCT) scans of specimens to uncover
internal features and natural history information without incurring
physical damage via dissection. Unlike hard-tissue imaging, diceCT
datasets are currently limited to a few individual specimens and
taxonomically underrepresented. To initiate best practices for diceCT in
a non-model group, we outline a guide for staining and high-throughput
µCT scanning in snakes. We scanned the entire body and one region of
interest (i.e., head) for 23 specimens representing 23 species from the
clades Aniliidae, Dipsadinae, Colubrinae, Elapidae, Lamprophiidae and
Viperidae. We generated 82 scans that include 1.25% Lugols iodine
stained (soft tissue) and unstained (skeletal) data for each specimen.
We found that duration of optimal staining time increased linearly with
body size; head radius was the best indicator. Post-reconstruction of
scans, optimal staining was evident by evenly distributed grayscale
values and clear differentiation among soft-tissue anatomy. Under and
over stained specimens produced poor contrast among soft-tissues, which
was often exacerbated by user bias during “digital dissections” (i.e.,
segmentation). Regardless, all scans produced usable data from which we
assessed a range of downstream analytical applications within ecology
and evolution (e.g., predator-prey interactions, life history, and
morphological evolution). Ethanol de-staining reversed the known effects
of iodine on the exterior appearance of physical specimens, but required
substantially more time than reported for other de-staining methods. We
discuss the feasibility of implementing diceCT techniques for a new
user, including approximate financial and temporal commitments, required
facilities, and potential effects of staining on specimens. We present
the first high-throughput workflow for full-body skeletal and diceCT
scanning in snakes, which can be generalized to any elongate
vertebrates, and increases publicly available diceCT scans for reptiles
by an order of magnitude.