Morphologists have historically had to rely on destructive procedures to visualize the three‐dimensional (3‐D) anatomy of animals. More recently, however, non‐destructive techniques have come to the forefront. These include X‐ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard‐tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT‐based research is the use of chemical agents to render visible, and differentiate between, soft‐tissue structures in X‐ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine‐based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine‐based, contrast‐enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting‐edge applications of diffusible iodine‐based contrast‐enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.

The cochlea and semicircular canals (SCCs) of the inner ear are vital neurosensory devices. There are associations between the anatomy of these sensorineural structures, their function, and the function of related biological systems, for example, hearing ability, gaze stabilization, locomotor agility, and posture. The endosseous labyrinth is frequently used as a proxy to infer the performance of the hearing and vestibular systems, locomotor abilities, and ecology of extinct species. Such fossil inferences are often based on single specimens or even a single ear, representing an entire species. To address whether a single ear is representative of a population, we used geometric morphometrics to quantitatively assess the variation in shape and symmetry in a sample of endosseous labyrinths of wild turkeys Meleagris gallopavo of southern Ohio. We predicted that ears would be symmetrical both within individuals and across the sample; that labyrinth shape and size would covary; that labyrinth shape would vary with the size of the brain, measured as width of the endocranium at the cerebellum; and that labyrinths would be morphologically integrated. To test these predictions, we microCT-scanned the heads of 26 cadaveric turkeys, digitally segmented their endosseous labyrinths in Avizo, and assigned 15 manual landmarks and 20 sliding semilandmarks to each digital model. Following Procrustes alignment, we conducted an analysis of bilateral symmetry, a Procrustes regression analysis for allometry and other covariates including side and replicate, and analyses of global integration and modularity. Based on Procrustes distances, no individual’s left and right ears were clearly different from each other. When comparing the ears of different specimens, statistically clear differences in shape were found in only 66 of more than 1,300 contrasts. Moreover, effects of both directional and fluctuating asymmetry were very small—generally, two orders of magnitude smaller than the variance explained by individual variation. Statistical tests disagreed on whether these asymmetric effects crossed the threshold of significance, possibly due to non-isotropic variation among landmarks. Regardless, labyrinths appeared to primarily vary in shape symmetrically. Neither labyrinth size nor endocranial width was correlated with labyrinth shape, contrary to our expectations. Finally, labyrinths were found to be moderately integrated in a global sense, but four weakly separated modules—the three SCCs and cochlea—were recovered using a maximum-likelihood analysis. The results show that both fluctuating and directional asymmetry play a larger role in shape variation than expected—but nonetheless, endosseous labyrinths are symmetrical within individuals and at the level of the population, and their shape varies symmetrically. Thus, inferences about populations, and very possibly species, may be confidently made when only a single specimen, or even a single ear, is available for study.

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The treatment of vertebrate specimens with radio‐opaque substances to enhance soft‐tissue contrast in CT scans has revolutionized morphological analysis. DiceCT has produced spectacular results and involves immersing specimens in Lugol's iodine. A shortcoming of diceCT is that diffusion (the “d” in “diceCT”) can take days, weeks, or months in large, intact, unskinned specimens. Moreover, long diffusion times can cause marked shrinkage. Alternatively, our team has developed spiceCT, which involves perfusing specimens with Lugol's iodine, yielding excellent results—literally within hours. The vascular system of thawed, unfixed, unskinned specimens (mostly birds and reptiles thus far) is cannulated, and then hypertonic (2.5 or 5%) aqueous Lugol's iodine is injected with a syringe. The solution perfuses well, easily filling capillary beds. Perfusion can be visually monitored in key areas, as well as tactilely via syringe pressure. Staining is rapid, and specimens can be scanned immediately, yielding same‐day results. There is no time for shrinkage, which is a well‐known problem for diceCT specimens immersed in iodine for long periods. The absence of prior fixation shortens processing time and also opens new avenues for final storage in that the specimen can be fixed, refrozen, or even skeletonized. Perfusion rather than diffusion also allows targeting of tissues—selective perfusion (the “sp” of “spiceCT”)—by injecting their vascular supply. One shortcoming of spiceCT is that iodine is too large to cross the blood‐brain barrier (BBB), even in cadaveric specimens. Our team is currently exploring ways to overcome this problem, experimenting with adding EDTA to the Lugol's injection medium to disrupt the cadherins forming the tight junctions of the endothelial cells of the cerebral vasculature. Initial results are promising. SpiceCT is intended to supplement, not replace, diceCT in the toolkit of morphologists.Support or Funding InformationUS National Science Foundation (IOB‐0517257, IOS‐1050154, IOS‐1456503)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Although the visual system of crocodylians has attracted interest regarding optical parameters and retinal anatomy, fundamental questions remain about the allometry of the eyeball and whether such scaling is the same across all crown groups of crocodylians. In addition, anatomy and identities of adnexal soft tissues that interact with the visual system are not well understood in many cases. We used contrast‐enhancing iodine stain and high‐resolution micro‐computed tomography to assess the anatomy of orbital soft tissues, including extraocular muscles and glands, in crocodylians. We also used regression analysis to estimate the allometric relationship between the bony orbit and eyeball across Alligator mississippiensis and Crocodylus niloticus for the first time. Results revealed tight, negatively allometric relationships between the bony orbit and eyeball. Notably, the eyes of C. niloticus were larger for a given orbit size than the eyes of A. mississippiensis, although the slope of the relationship was no different between these two crown crocodylian groups. Among the findings from our anatomical study, new details were uncovered about the homologies of muscles of the abducens complex. In particular, M. rectus lateralis of crocodylians is revealed to have a more complex form than previously appreciated, being adhered to the tendon of the nictitating membrane, which may be apomorphic for Crocodylia. Our calculation of the orbit‐eyeball allometric relationship and study of the adnexal soft tissues of the crocodylian visual system, in combination with previous work by other teams in other crown saurian clades, is a critical, formerly missing, piece in the Extant Phylogenetic Bracket for restoring the visual apparatus of extinct crocodyliforms and other archosauriform groups.