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1. DiceCT for Fishes: Recommendations for pairing iodine contrast agents with μCT to visualize soft tissues in fishes

   https://doi.org/10.1111/jfb.15320  

   Kolmann, Matthew A.; Nagesan, Ramon S.; Andrews, James V.; Borstein, Samuel; Figueroa, Rodrigo Tinoco; Singer, Randal; Friedman, Matt; López‐Fernández, Hernán (January 2023, Journal of Fish Biology)

   Computed tomography (CT) scanning and other high-throughput 3D visualization tools are transforming the ways we study morphology, ecology, and evolutionary biology research beyond generating vast digital repositories of anatomical data. Contrast-enhanced chemical staining methods, which render soft tissues radio-opaque when coupled with CT scanning, encompass several approaches that are growing in popularity and versatility. Of these, the various diceCT techniques that use an iodine-based solution like Lugol's have provided access to an array of morphological datasets spanning extant vertebrate lineages. This contribution outlines straightforward means for applying diceCT techniques to preserved museum specimens of cartilaginous and bony fishes, collectively representing half of vertebrate species diversity. We contrast the benefits of using either aqueous or ethylic Lugol's solutions and report few differences between these methods with respect to the time required to achieve optimal tissue contrast. We also explore differences in minimum stain duration required for different body sizes and shapes and provide recommendations for staining specimens individually or in small batches. As reported by earlier studies, we note a decrease in pH during staining with either aqueous or ethylic Lugol's. However, we could not replicate the drastic declines in pH reported elsewhere. We provide recommendations for researchers and collections staff on how to incorporate diceCT into existing curatorial practices, while offsetting risk to specimens. Finally, we outline how diceCT with Lugol's can aid ichthyologists of all kinds in visualizing anatomical structures of interest: from brains and gizzards to gas bladders and pharyngeal jaw muscles. 

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2. Ethanol-preserved eyes provide ocular and retinal predictors of natural morphological conditions in scallops: a case study with Argopecten irradians (Bivalvia: Pectinidae)

   https://doi.org/10.1093/mollus/eyae044  

   Audino, Jorge A; Quinlan, Gerald; Ordas, Ceren; Serb, Jeanne M (October 2024, Journal of Molluscan Studies)

   usp.br Ethanol-preserved specimens represent one of the most common resources for biological research. However, little is known of how ethanol preservation may change tissue morphology and impact the interpretation of trait quantification in structures, such as eyes. While scallop eyes are an interesting system for investigating eye evolution and visual adaptations, cross-species comparisons mainly depend on museum specimens. Therefore, to test whether ethanol-preserved specimens serve as accurate indicators of natural eye morphology, we investigated the effects of preservation on selected traits, such as eye and pupil diameter, in the scallop Argopecten irradians . We also compared ethanol-preserved eyes to paraformaldehyde (PFA)-fixed eyes to investigate possible impacts on retinal morphology. Our results demonstrate that eye size does not change with short-term preservation, whereas pupil size becomes significantly larger, likely due to the contraction of actin fibers during dehydration. When comparing measurements among eyes and treatments, eye size correlates to pupil size, but is not correlated to body size. We found that ethanol-preserved eyes provide close estimates of retinal traits, with similar photoreceptor spacing distance and number of photoreceptor cells, compared to samples fixed in PFA. These findings might also be applicable in the context of other mollusks, especially bivalves and gastropods, with delicate visual systems. Our study provides evidence that ethanol- preserved eyes exhibit tissue-specific differences that should be acknowledged in morphological studies. For example, pupil size should be investigated while accounting for post-preservation effects. Other traits, such as lens shape, are inconsistent and severely impacted by preservation. Finally, eye size and some photoreceptor cell measurements can be helpful to describe natural morphology. 

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3. A comparison of diceCT and histology for determination of nasal epithelial type

   https://doi.org/10.7717/peerj.12261  

   Smith, Timothy D.; Corbin, Hayley M.; King, Scot E.; Bhatnagar, Kunwar P.; DeLeon, Valerie B. (January 2021, PeerJ)

   Diffusible iodine-based contrast-enhanced computed tomography (diceCT) has emerged as a viable tool for discriminating soft tissues in serial CT slices, which can then be used for three-dimensional analysis. This technique has some potential to supplant histology as a tool for identification of body tissues. Here, we studied the head of an adult fruit bat ( Cynopterus sphinx ) and a late fetal vampire bat ( Desmodus rotundus ) using diceCT and µCT. Subsequently, we decalcified, serially sectioned and stained the same heads. The two CT volumes were rotated so that the sectional plane of the slice series closely matched that of histological sections, yielding the ideal opportunity to relate CT observations to corresponding histology. Olfactory epithelium is typically thicker, on average, than respiratory epithelium in both bats. Thus, one investigator (SK), blind to the histological sections, examined the diceCT slice series for both bats and annotated changes in thickness of epithelium on the first ethmoturbinal (ET I), the roof of the nasal fossa, and the nasal septum. A second trial was conducted with an added criterion: radioopacity of the lamina propria as an indicator of Bowman’s glands. Then, a second investigator (TS) annotated images of matching histological sections based on microscopic observation of epithelial type, and transferred these annotations to matching CT slices. Measurements of slices annotated according to changes in epithelial thickness alone closely track measurements of slices based on histologically-informed annotations; matching histological sections confirm blind annotations were effective based on epithelial thickness alone, except for a patch of unusually thick non-OE, mistaken for OE in one of the specimens. When characteristics of the lamina propria were added in the second trial, the blind annotations excluded the thick non-OE. Moreover, in the fetal bat the use of evidence for Bowman’s glands improved detection of olfactory mucosa, perhaps because the epithelium itself was thin enough at its margins to escape detection. We conclude that diceCT can by itself be highly effective in identifying distribution of OE, especially where observations are confirmed by histology from at least one specimen of the species. Our findings also establish that iodine staining, followed by stain removal, does not interfere with subsequent histological staining of the same specimen. 

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4. Effects of freezing and short‐term fixation on muscle mass, volume, and density

   https://doi.org/10.1002/ar.24639  

   Leonard, Kaitlyn C; Worden, Nikole; Boettcher, Marissa L; Dickinson, Edwin; Hartstone‐Rose, Adam (January 2022, The Anatomical Record)

   Abstract Preventing postmortem deterioration of soft‐tissues is an important requisite of anatomical research. In order to provide corrections for potential myological distortions, this study quantifies the acute effects of freezing, formalin fixation and ethanol storage using muscles from (n = 46) rabbits (Oryctolagus cuniculus). Bilateral dissections of specific muscles were performed and each side was assigned to a different preparation group (fresh, formalin fixation only, fixation followed by short duration ethanol storage, and freezing once or twice). We demonstrate that short‐term freezing at −20C and thawing have no significant effect on muscle mass, volume, and density while short‐term formalin fixation and ethanol storage significantly reduces mass and volume (density remains relatively constant.) Although freezing may have less of an effect on the gross morphometric characteristics of the musculature than ethanol storage, slow freezing damages muscle microanatomy, and therefore, faster freezing and other modes of preservation such as formalin fixation and ethanol storage may be preferable. Based on our results, we derived the following correction factors for each preparation: the mass of specimens stored in 70% ethanol should be multiplied by 1.69 to approximate fresh muscle mass, and specimens fixed in 10% formalin multiplied by 1.32. Although not significant, specimens frozen‐once were slightly less massive and could be multiplied by 1.03 (frozen‐twice ×1.09). The volumetric corrections are: ethanol 1.64; 10% formalin 1.32; frozen‐once 1.03; frozen‐twice 1.10. While the density of ethanol preserved specimens is slightly less than that of fresh ones (correction: 1.03), those preserved in formalin and frozen maintain nearly the same density. 

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5. Adaptation and innovation in darter fish cranial musculature (Etheostomatinae: Percidae): insights from diceCT

   https://doi.org/10.1093/zoolinnean/zlae135  

   Arbour, J H; Ramazan, S; Clark, S (November 2024, Zoological Journal of the Linnean Society)

   Abstract Fish skulls are often highly kinetic, with multiple linkage and lever systems powered by a diverse suite of muscles. Comparative analysis of the evolution of soft-tissue structures in the fish skull is often limited under traditional approaches, while new imaging techniques like diceCT (diffusible iodine-based contrast-enhanced computed tomography) allow for high-resolution imaging of muscles in situ. Darters (Percidae: Etheostomatinae) are a diminutive and species-rich clade of lotic freshwater fishes, which show diverse head shapes believed to be associated with different foraging strategies. We used diceCT to sample all major cranial adductors and abductors responsible for movement of the jaw, hyoid, operculum, and suspensorium from 29 species. We applied comparative phylogenetic approaches to analyse the evolutionary trends in muscle size across the clade. We found two major patterns: (i) darter cranial muscles show fundamental trade-offs relating to investment in musculature, as well as buccal expansion vs. biting attributes; early divergence in muscle size appears to be associated with shifts in habitat use and foraging; (ii) darter adductor mandibulae show high variation in architecture (fibre orientation, divisions). This study highlights how new imaging techniques can provide novel insights into the anatomy of even well-sampled/represented clades. 

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