Evaluation of pore-fracture microstructure of gypsum rock fragments using micro-CT.

This study utilized X-ray micro-computed tomography (micro-CT) to investigate weathered gypsum rocks which can or do serve as a rock substrate for endolithic organisms, focusing on their internal pore-fracture microstructure, estimating porosity, and quantitative comparison between various samples. Examining sections and reconstructed 3D models provides a more detailed insight into the overall structural conditions within rock fragments and the interconnectivity in pore networks, surpassing the limitations of analyzing individual 2D images. Results revealed diverse gypsum forms, cavities, fractures, and secondary features influenced by weathering. Using deep learning segmentation based on the U-Net models within the Dragonfly software enabled to identify and visualize the porous systems and determinate void space which was used to calculate porosity. This approach allowed to describe what type of microstructures and cavities is responsible for the porous spaces in different gypsum samples. A set of quantitative analysis of the detected void and modeled networks provided a needed information about the development of the pore system, connectivity, and pore size distribution. Comparison with mercury intrusion porosimetry showed that both methods consider different populations of pores. In our case, micro-CT typically detects larger pores (> 10 µm) which is related to the effective resolution of the scanned images. Still, micro-CT demonstrated to be an efficient tool in examining the internal microstructures of weathered gypsum rocks, with promising implications particularly in geobiology and microbiology for the characterization of lithic habitats.

Gypsum endolithic phototrophs under moderate climate (Southern Sicily): their diversity and pigment composition.

In this study, we used microscopic, spectroscopic, and molecular analysis to characterize endolithic colonization in gypsum (selenites and white crystalline gypsum) from several sites in Sicily. Our results showed that the dominant microorganisms in these environments are cyanobacteria, including: Chroococcidiopsis sp., Gloeocapsopsis pleurocapsoides, Gloeocapsa compacta, and Nostoc sp., as well as orange pigmented green microalgae from the Stephanospherinia clade. Single cell and filament sequencing coupled with 16S rRNA amplicon metagenomic profiling provided new insights into the phylogenetic and taxonomic diversity of the endolithic cyanobacteria. These organisms form differently pigmented zones within the gypsum. Our metagenomic profiling also showed differences in the taxonomic composition of endoliths in different gypsum varieties. Raman spectroscopy revealed that carotenoids were the most common pigments present in the samples. Other pigments such as gloeocapsin and scytonemin were also detected in the near-surface areas, suggesting that they play a significant role in the biology of endoliths in this environment. These pigments can be used as biomarkers for basic taxonomic identification, especially in case of cyanobacteria. The findings of this study provide new insights into the diversity and distribution of phototrophic microorganisms and their pigments in gypsum in Southern Sicily. Furthemore, this study highlights the complex nature of endolithic ecosystems and the effects of gypsum varieties on these communities, providing additional information on the general bioreceptivity of these environments.

The first description of dermal armour in snakes.

Osteoderms, also called dermal armour, often play a role in predator defence. The presence of osteoderms is highly irregularly distributed across the squamate phylogeny and they have not been found in snakes. In this study, we searched for candidate snake species that would benefit from such armour to protect their body, focusing primarily on fossorial species with defensive tail displays. We examined the tail morphology of 27 snake species from different families using micro-computed tomography (µCT) and micro- radiography. We discovered dermal armour in four species of sand boas (Erycidae) that also feature enlarged and highly modified caudal vertebrae. This is the first description of dermal armour in snakes. Ancestral state reconstructions revealed that osteoderms likely evolved once or multiple times in Erycidae. We have not found osteoderms in any other examined snake species. Nevertheless, similar structures are known from unrelated squamate clades, such as gerrhosaurids and geckos. This supports the idea of underlying deep developmental homology. We propose the hypothesis that osteoderms protect sand boas like the "brigandine armour" of medieval warriors. We interpret it as another component of the sand boas' rich defence strategy.

Determinate growth is predominant and likely ancestral in squamate reptiles.

Body growth is typically thought to be indeterminate in ectothermic vertebrates. Indeed, until recently, this growth pattern was considered to be ubiquitous in ectotherms. Our recent observations of a complete growth plate cartilage (GPC) resorption, a reliable indicator of arrested skeletal growth, in many species of lizards clearly reject the ubiquity of indeterminate growth in reptiles and raise the question about the ancestral state of the growth pattern. Using X-ray micro-computed tomography (µCT), here we examined GPCs of long bones in three basally branching clades of squamate reptiles, namely in Gekkota, Scincoidea and Lacertoidea. A complete loss of GPC, indicating skeletal growth arrest, was the predominant finding. Using a dataset of 164 species representing all major clades of lizards and the tuataras, we traced the evolution of determinate growth on the phylogenetic tree of Lepidosauria. The reconstruction of character states suggests that determinate growth is ancestral for the squamate reptiles (Squamata) and remains common in the majority of lizard lineages, while extended (potentially indeterminate) adult growth evolved several times within squamates. Although traditionally associated with endotherms, determinate growth is coupled with ectothermy in this lineage. These findings combined with existing literature suggest that determinate growth predominates in both extant and extinct amniotes.

Universality of indeterminate growth in lizards rejected: the micro-CT reveals contrasting timing of growth cartilage persistence in iguanas, agamas, and chameleons.

Squamate reptiles are considered to exhibit indeterminate growth. Nevertheless, current literature disputes the available definitions of this growth type, presents new theoretical models, and questions its universality in cold-blooded vertebrates. We have followed up on our previous research employing micro-CT to explore growth plate cartilage (GPC) in the epiphysis of long bones, which is responsible for longitudinal skeletal growth by the endochondral ossification process. We focused on numerous and highly diversified group of the Iguania clade comprising Acrodonta (agamas and chameleons) and Pleurodonta ("iguanas"). We recorded the absence of GPC in most of the examined adult Pleurodonta specimens and interpret it as an irreversible arrest of skeletal growth. This finding clearly rejects the universality of indeterminate growth in lizards. On the other hand, we found apparent GPC preservation in most of the adult specimens belonging to Acrodonta. This suggests a preserved ability to continue body growth throughout most of their life. We discuss the uncovered disparity between Acrodonta and Pleurodonta and emphasize the importance of GPC degradation timing.

Imaging of Mouse Brain Fixated in Ethanol in Micro-CT.

Micro-CT imaging is a well-established morphological method for the visualization of animal models. We used ethanol fixation of the mouse brains to perform high-resolution micro-CT scans showing in great details brain grey and white matters. It was possible to identify more than 50 neuroanatomical structures on the 5 selected coronal sections. Among white matter structures, we identified fornix, medial lemniscus, crossed tectospinal pathway, mammillothalamic tract, and the sensory root of the trigeminal ganglion. Among grey matter structures, we identified basal nuclei, habenular complex, thalamic nuclei, amygdala, subparts of hippocampal formation, superior colliculi, Edinger-Westphal nucleus, and others. We suggest that micro-CT of the mouse brain could be used for neurohistological lesions evaluation as an alternative to classical neurohistology because it does not destroy brain tissue.

Ethanol fixation method for heart and lung imaging in micro-CT.

PURPOSE: The soft tissue imaging in micro-CT remains challenging due to its low intrinsic contrast. The aim of this study was to create a simple staining method omitting the usage of contrast agents for ex vivo soft tissue imaging in micro-CT. MATERIALS AND METHODS: Hearts and lungs from 30 mice were used. Twenty-seven organs were either fixed in 97% or 50% ethanol solution or in a series of ascending ethanol concentrations. Images were acquired after 72, 168 and 336 h on a custom-built micro-CT machine and compared to scans of three native samples. RESULTS: Ethanol provided contrast enhancement in all evaluated fixations. Fixation in 97% ethanol resulted in contrast enhancement after 72 h; however, it caused hardening of the samples. Fixation in 50% ethanol provided contrast enhancement after 336 h, with milder hardening, compared to the 97% ethanol fixation, but the visualization of details was worse. The fixation in a series of ascending ethanol concentrations provided the most satisfactory results; all organs were visualized in great detail without tissue damage. CONCLUSIONS: Simple ethanol fixation improves the tissue contrast enhancement in micro-CT. The best results can be obtained with fixation of the soft tissue samples in a series of ascending ethanol concentrations.

Non-destructive imaging of fragments of historical beeswax seals using high-contrast X-ray micro-radiography and micro-tomography with large area photon-counting detector array.

Historical beeswax seals are unique cultural heritage objects. Unfortunately, a number of historical sealing waxes show a porous structure with a strong tendency to stratification and embrittlement, which makes these objects extremely prone to mechanical damage. The understanding of beeswax degradation processes therefore plays an important role in the preservation and consequent treatment of these objects. Conventional methods applied for the investigation of beeswax materials (e.g. gas chromatography) are of a destructive nature or bring only limited information about the sample surface (microscopic techniques). Considering practical limitations of conventional methods and ethical difficulties connected with the sampling of the historical material, radiation imaging methods such as X-ray micro-tomography presents a promising non-destructive tool for the onward scientific research in this field. In this contribution, we present the application of high-contrast X-ray micro-radiography and micro-tomography for the investigation of beeswax seal fragments. The method is based on the application of the large area photon-counting detector recently developed at our institute. The detector combines the advantages of single-photon counting technology with a large field of view. The method, consequently, enables imaging of relatively large objects with high geometrical magnification. In the reconstructed micro-tomographies of investigated historical beeswax seals, we are able to reveal morphological structures such as stratification, micro-cavities and micro-fractures with spatial resolution down to 5µm non-destructively and with high imaging quality. The presented work therefore demonstrates that a combination of state-of-the-art hybrid pixel semiconductor detectors and currently available micro-focus x-ray sources makes it possible to apply X-ray micro-radiography and micro-tomography as a valuable non-destructive tool for volumetric beeswax seal morphological studies.

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector.

Using dedicated contrast agents high-quality X-ray imaging of soft tissue structures with isotropic micrometre resolution has become feasible. This technique is frequently titled as virtual histology as it allows production of slices of tissue without destroying the sample. The use of contrast agents is, however, often an irreversible time-consuming procedure and despite the non-destructive principle of X-ray imaging, the sample is usually no longer usable for other research methods. In this work we present the application of recently developed large-area photon counting detector for high resolution X-ray micro-radiography and micro-tomography of whole ex-vivo ethanol-preserved mouse organs. The photon counting detectors provide dark-current-free quantum-counting operation enabling acquisition of data with virtually unlimited contrast-to-noise ratio (CNR). Thanks to the very high CNR even ethanol-only preserved soft-tissue samples without addition of any contrast agent can be visualized in great detail. As ethanol preservation is one of the standard steps of tissue fixation for histology, the presented method can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft-tissue visualisation.