Dataset Information

Chemical effects of diceCT staining protocols on fluid-preserved avian specimens.

ABSTRACT: Diffusible iodine-based contrast-enhanced computed tomography (diceCT) techniques allow visualization of soft tissues of fluid-preserved specimens in three dimensions without dissection or histology. Two popular diceCT stains, iodine-potassium iodide (I2KI) dissolved in water and elemental iodine (I2) dissolved in 100% ethanol (EtOH), yield striking results. Despite the widespread use of these stains in clinical and biological fields, the molecular mechanisms that result in color change and radiopacity attributed to iodine staining are poorly understood. Requests to apply these stains to anatomical specimens preserved in natural history museums are increasing, yet curators have little information about the potential for degradation of treated specimens. To assess the molecular effects of iodine staining on typical museum specimens, we compared the two popular stains and two relatively unexplored stains (I2KI in 70% EtOH, I2 in 70% EtOH). House sparrows (Passer domesticus) were collected and preserved under uniform conditions following standard museum protocols, and each was then subjected to one of the stains. Results show that the three ethanol-based stains worked equally well (producing fully stained, life-like, publication quality scans) but in different timeframes (five, six, or eight weeks). The specimen in I2KI in water became degraded in physical condition, including developing flexible, demineralized bones. The ethanol-based methods also resulted in some demineralization but less than the water-based stain. The pH of the water-based stain was notably acidic compared to the water used as solvent in the stain. Our molecular analyses indicate that whereas none of the stains resulted in unacceptable levels of protein degradation, the bones of a specimen stained with I2KI in water demineralized throughout the staining process. We conclude that staining with I2KI or elemental I2 in 70% EtOH can yield high-quality soft-tissue visualization in a timeframe that is similar to that of better-known iodine-based stains, with lower risk of negative impacts on specimen condition.

SUBMITTER: Early CM

PROVIDER: S-EPMC7500670 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Chemical effects of diceCT staining protocols on fluid-preserved avian specimens.

Early Catherine M CM Morhardt Ashley C AC Cleland Timothy P TP Milensky Christopher M CM Kavich Gwénaëlle M GM James Helen F HF

PloS one 20200918 9

Diffusible iodine-based contrast-enhanced computed tomography (diceCT) techniques allow visualization of soft tissues of fluid-preserved specimens in three dimensions without dissection or histology. Two popular diceCT stains, iodine-potassium iodide (I2KI) dissolved in water and elemental iodine (I2) dissolved in 100% ethanol (EtOH), yield striking results. Despite the widespread use of these stains in clinical and biological fields, the molecular mechanisms that result in color change and radi ...[more]

PMID: 32946473

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Project description:Diffusible iodine-based contrast-enhanced computed tomography (diceCT) visualizes soft tissue from micro-CT (µCT) scans of specimens to uncover internal features and natural history information without incurring physical damage via dissection. Unlike hard-tissue imaging, taxonomic sampling within diceCT datasets is currently limited. To initiate best practices for diceCT in a nonmodel 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% Lugol's 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. Postreconstruction 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 destaining reversed the known effects of iodine on the exterior appearance of physical specimens, but required substantially more time than reported for other destaining 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.

Project description:Amniotes feature two principal visual processing systems: the tectofugal and thalamofugal pathways. In most mammals, the thalamofugal pathway predominates, routing retinal afferents through the dorsolateral geniculate complex to the visual cortex. In most birds, the thalamofugal pathway often plays the lesser role with retinal afferents projecting to the principal optic thalami, a complex of several nuclei that resides in the dorsal thalamus. This thalamic complex sends projections to a forebrain structure called the Wulst, the terminus of the thalamofugal visual system. The thalamofugal pathway in birds serves many functions such as pattern discrimination, spatial memory, and navigation/migration. A comprehensive analysis of avian species has unveiled diverse subdivisions within the thalamic and forebrain structures, contingent on species, age, and techniques utilized. In this study, we documented the thalamofugal system in three dimensions by integrating histological and contrast-enhanced computed tomography imaging of the avian brain. Sections of two-week-old chick brains were cut in either coronal, sagittal, or horizontal planes and stained with Nissl and either Gallyas silver or Luxol Fast Blue. The thalamic principal optic complex and pallial Wulst were subdivided on the basis of cell and fiber density. Additionally, we utilized the technique of diffusible iodine-based contrast-enhanced computed tomography (diceCT) on a 5-week-old chick brain, and right eyeball. By merging diceCT data, stained histological sections, and information from the existing literature, a comprehensive three-dimensional model of the avian thalamofugal pathway was constructed. The use of a 3D model provides a clearer understanding of the structural and spatial organization of the thalamofugal system. The ability to integrate histochemical sections with diceCT 3D modeling is critical to better understanding the anatomical and physiologic organization of complex pathways such as the thalamofugal visual system.

Dataset Information

Chemical effects of diceCT staining protocols on fluid-preserved avian specimens.

Publications

Chemical effects of diceCT staining protocols on fluid-preserved avian specimens.

Similar Datasets

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