Microscopic dual-energy CT (microDECT): a flexible tool for multichannel ex vivo 3D imaging of biological specimens.

Dual-energy computed tomography (DECT) uses two different x-ray energy spectra in order to differentiate between tissues, materials or elements in a single sample or patient. DECT is becoming increasingly popular in clinical imaging and preclinical in vivo imaging of small animal models, but there have been only very few reports on ex vivo DECT of biological samples at microscopic resolutions. The present study has three main aims. First, we explore the potential of microscopic DECT (microDECT) for delivering isotropic multichannel 3D images of fixed biological samples with standard commercial laboratory-based microCT setups at spatial resolutions reaching below 10 µm. Second, we aim for retaining the maximum image resolution and quality during the material decomposition. Third, we want to test the suitability for microDECT imaging of different contrast agents currently used for ex vivo staining of biological samples. To address these aims, we used microCT scans of four different samples stained with x-ray dense contrast agents. MicroDECT scans were acquired with five different commercial microCT scanners from four companies. We present a detailed description of the microDECT workflow, including sample preparation, image acquisition, image processing and postreconstruction material decomposition, which may serve as practical guide for applying microDECT. The MATLAB script (The Mathworks Inc., Natick, MA, USA) used for material decomposition (including a graphical user interface) is provided as a supplement to this paper (https://github.com/microDECT/DECTDec). In general, the presented microDECT workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in the separate material fractions after basis material decomposition. In addition to decomposition of mineralized tissues (inherent sample contrast) and stained soft tissues, we present a case of double labelling of different soft tissues with subsequent material decomposition. We conclude that, in contrast to in vivo DECT examinations, small ex vivo specimens offer some clear advantages regarding technical parameters of the microCT setup and the use of contrast agents. These include a higher flexibility in source peak voltages and x-ray filters, a lower degree of beam hardening due to small sample size, the lack of restriction to nontoxic contrast agents and the lack of a limit in exposure time and radiation dose. We argue that microDECT, because of its flexibility combined with already established contrast agents and the vast number of currently unexploited stains, will in future represent an important technique for various applications in biological research.

Light and transmission electron microscopy of the dorsal lingual epithelium of Pelusios castaneus (Pleurodira, Chelidae) with special respect to its feeding mechanics.

The ultrastructure of the dorsal lingual epithelium of the semi-aquatic West African mud turtle, Pelusios castaneus, is described. Our goal is to give additional information to previous studies of this species such as feeding pattern analysis and gross morphology. Tissue specimens were fixed in modified Karnovsky solution followed by osmium tetroxide, embedded in epoxy resin and observed using light and transmission electron microscopy. The dorsal tongue surface is covered with moderate papillae, which are coated by a stratified epithelium overlying a connective tissue core. Two epithelial regions can be differentiated, although differences are not very obvious: the apical area, where granular cells are more abundant than mucus cells, and the lateral area, where cell distribution is opposite. Within the epithelium, different layers are discernable on the basis of the cells' organelles, corresponding with a process of cell maturation and formation of different granules. These results together with data of previous studies of this species show that the ultrastructure of the lingual epithelium is similar to other turtles adapted to semi-aquatic environments; functional and morphological data indicate a generalist, being well but not highly adapted to feeding in an aquatic environment.

Morphology and function of the feeding apparatus of Pelusios castaneus (Chelonia; Pleurodira).

Feeding mechanics of vertebrates depend on physical constraints of the surrounding media, water or air. Such functions are inseparably combined with form. The aim of this study is to show this linkage for the pleurodiran freshwater turtle Pelusios castaneus and, additionally, to point out the major functional and biomechanical distinctions between aquatic and terrestrial feeding turtles as well as several intermediate forms. Gross morphological investigations of skull, hyoid, tongue, and connected musculature, as well as scanning electron microscopy of the tongue surface, show typical features of an aquatic feeder, e.g., strongly developed hyoid apparatus vs. a small tongue with only moderate papillae, and massive jaw and hyoid musculature. Additionally, the special function of the esophagus during feeding is investigated to elucidate the problems of a bidirectional feeder. The esophagus is highly distensible in order to store the excess water sucked in during feeding until the prey is fixed by the jaws. The distension is probably achieved by a coincidence of active (branchial horn) as well as passive (water) components. P. castaneus is a feeding generalist, and is well adapted to the aquatic medium in terms of its functional as well as morphological features.

Fine structure of the dorsal lingual epithelium of Trachemys scripta elegans (Chelonia: Emydidae).

BACKGROUND: Turtles are adapted to different environments, such as freshwater, marine, and terrestrial habitats. Examination of histological and ultrastructural features of the dorsal lingual epithelium of the red-eared turtle, Trachemys scripta elegans, and comparison of the results with those of other turtles should elucidate the relationship between the morphology of tongues as well as the fine structure of lingual epithelia and chelonian feeding mechanisms. METHODS: Light microscopical (LM) and scanning (SEM) and transmission (TEM) electron microscopical methods were used. RESULTS: SEM revealed a distribution of lingual papillae all over the dorsal tongue surface. Single epithelial cells can be discerned, with short microvilli on their surface. LM studies show differences within the stratified epithelium between the lateral and the apical side of the papillae. In TEM, these differences become more obvious; while the basal and deep intermediate layer is similar in both sides of the papillae, mucus granules begin to form at the edge of the superficial intermediate layer at the lateral side. Cells containing fine secretory granules are visible there, too. On the other hand, at the apical side, only fine-granule-containing cells are visible. CONCLUSIONS: This study indicates that the histology and ultrastructure of the lingual epithelium of Trachemys scripta elegans are similar to that of other turtles adapted to freshwater environments but differ from those of turtles living in marine or terrestrial habits. These differences can be explained in terms of the adaptation of turtles to their particular life circumstances.