No abstract

Background Given the need for descriptive and increasingly mechanistic morphological analyses, contrast‐enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro‐level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA‐enhanced microCT. Results Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. Conclusion These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

Sampling small benthic and lithophilic fish species in large rivers and lakes presents challenges not adequately addressed by conventional survey methods such as boat electrofishing and gill netting. The development of the Missouri trawl has helped to address these issues; however, our observations by scuba diving when using the Missouri trawl have revealed avoidance of the trawl by benthic fishes, especially in rocky substrates. Therefore, we equipped a Missouri trawl with a cathode-anode electrical array to facilitate capture by attracting and immobilizing fish. In 40 paired comparisons with a standard Missouri trawl in the upper Ohio River drainage of Pennsylvania, this electrified PSU trawl captured significantly more fish and species as well as more large fish. The PSU trawl also captured more species and more fish across habitats and rivers within the drainage. The PSU trawl is therefore a useful new device for sampling large-river benthic fish communities.

Abstract. Little information exists regarding the effects of mountain top removal/valley fill coal mining on stream fish populations in West Virginia and Kentucky. To address this knowledge gap, we conducted a study in cooperation with U.S. Environmental Protection Agency (USEPA) Region III to characterize the fish communities that exist in these regions and to evaluate the effects of these mining operations on fish populations. During 1999-2000, fish assemblages were sampled in 58 sites in West Virginia and in 15 sites in Kentucky. Results from this sampling effort indicated that not enough reference (unmined) sites were included to adequately assess the potential effects of mountain top mining/valley fill operations on fish communities in the area. We found a strong relationship between stream size (as described by stream order) and the total number of fish species present that confounded the effects of mining. As a result, in Fall 2001, we sampled 13 sites in the Guyandotte River drainage, including eight sites in the Mud River that were classified as filled or filled/residential and five reference (unmined) sites in the Big Ugly. Both the number of species and the number of benthic species present were greater in the reference sites than in the filled sites in 2001. Water chemistry analysis revealed that five of the Mud River sites sampled in 2001 had detectable levels of selenium (9.5 -31.5 µg/l). Sites that were associated with valley fills that had detectable levels of selenium seemed to be more impaired than sites associated with valley fills that had no detectable levels of selenium. Clearly, careful site selection and a multiple year collecting regimen are needed to determine the effects of these mining operations on stream fish assemblages.

Acquiring three‐dimensional imaging of animal specimens utilized in genetic, developmental, and evolutionary studies has typically been based on microCT (bone and mineralized tissues) and magnetic resonance microscopy (soft and un‐mineralized tissues). However, recent advances in microCT imaging have allowed for 1–5 μm resolution for small biological specimens, while typical microMRI imaging can generally only produce data with a resolution of approximately 30–40 μm. In order to achieve high‐resolution 3D visualization of soft tissues, the use of various contrast agents have been proposed, including iodine, osmium, and phosphotungstic acid (PTA). We investigated protocols for using PTA to stain soft tissues of the head in embryonic and early postnatal mice to enhance visualization of soft tissues by microCT imaging. Protocols for mice between embryonic day 13.5 and postnatal day 7 have been developed that allow for the visualization, segmentation, quantification, and analysis of soft tissue structures. Stained specimens were mounted in a 50:50 mix of polyester and paraffin waxes within a small tube and scanned on the GE v|tome|x L300 scanner in the Center for Quantitative Imaging at Pennsylvania State University with voxel dimensions between 1 and 10 μm, depending on specimen size. Multiple soft tissue structures, including the eye, inner ear, cartilage, muscle, glandular tissue, and brain, are easily differentiated in the resultant scans. Images of PTA‐stained specimens can be superimposed with microCT images of the same specimen before PTA staining to enable the study of relationships between hard and soft tissues in the same individual. This approach to both 2D and 3D visualization of soft tissues in murine models has immediate implications for understanding developmental processes and integration between hard and soft tissues of the cranium.Support or Funding InformationNational Science Foundation Grant BCS‐1731909, and NIDCR R01DE027677 and R01 DE022988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.