Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

X-ray phase contrast tomography for the investigation of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. Pre-clinical studies drive the development of animal models that well mimic ALS disorder and enable both the dissection of disease processes and an early assessment of therapy efficacy. A comprehensive knowledge of neuronal and vascular lesions in the brain and spinal cord is an essential factor to understand the development of the disease. Spatial resolution and bidimensional imaging are important drawbacks limiting current neuroimaging tools, while neuropathology relies on protocols that may alter tissue chemistry and structure. In contrast, recent ex vivo studies in mice demonstrated that X-ray phase-contrast tomography enables study of the 3D distribution of both vasculature and neuronal networks, without sample sectioning or use of staining. Here we present our findings on ex vivo SOD1G93A ALS mice spinal cord at a micrometric scale. An unprecedented direct quantification of neuro-vascular alterations at different stages of the disease is shown.

Exploring Alzheimer's disease mouse brain through X-ray phase contrast tomography: From the cell to the organ.

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aß) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aß plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/µm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

Periodically structured X-ray waveguides.

The properties of X-ray vacuum-gap waveguides (WGs) with additional periodic structure on one of the reflecting walls are studied. Theoretical considerations, numerical simulations and experimental results confirm that the periodic structure imposes additional conditions on efficient propagation of the electromagnetic field along the WGs. The transmission is maximum for guided modes that possess sufficient phase synchronism with the periodic structure (here called `super-resonances'). The field inside the WGs is essentially given at low incidence angle by the fundamental mode strongly coupled with the corresponding phased-matched mode. Both the simulated and the experimental diffraction patterns show in the far field that propagation takes place essentially only for low incidence angles, confirming the mode filtering properties of the structured X-ray waveguides.

A three-image algorithm for hard x-ray grating interferometry.

A three-image method to extract absorption, refraction and scattering information for hard x-ray grating interferometry is presented. The method comprises a post-processing approach alternative to the conventional phase stepping procedure and is inspired by a similar three-image technique developed for analyzer-based x-ray imaging. Results obtained with this algorithm are quantitatively comparable with phase-stepping. This method can be further extended to samples with negligible scattering, where only two images are needed to separate absorption and refraction signal. Thanks to the limited number of images required, this technique is a viable route to bio-compatible imaging with x-ray grating interferometer. In addition our method elucidates and strengthens the formal and practical analogies between grating interferometry and the (non-interferometric) diffraction enhanced imaging technique.

Human Pineal Gland Involutionary Process: New Findings.

In this work, we report preliminary results about the involution of the human pineal gland involution. The detailed analysis of pineal structure was done on autopsy material of 77 persons in age 27-96 using x-ray phase-contrast tomography, histology, and immunohistochemistry. Our study suggests that the pineal gland alteration in older adults may be more profound than has been reported to date. We identified and described a new form of pineal gland involution that eventually led to the total degradation of the pineal gland. To our knowledge, this study is the first to report on the complete replacement of pineal gland parenchyma with connective tissue in older adults.

Three-Dimensional Study of Polymer Composite Destruction in the Early Stages.

The investigation of destruction processes in composite materials is a current problem for their structural application and the improvement of their functional properties. This work aimed to visualize structural changes induced in layered carbon fiber reinforced plastics (CFRP) with the help of synchrotron X-ray microtomography. This article presents the details of destructive processes in the early stages of the deformation of reinforced polymers under uniaxial stretching, investigated at the micro level. Individual structural elements of the composite-filaments, parallel fiber bundles, the nonuniformity of the polymer binder distribution, and continuity defects-were observed under an external load. We have considered the influence of the material architecture and technological defects on fracture evolution in cross-ply and quasi-isotropic fiber-reinforced plastics. The results indicate the sequence of irreversible structural changes before the destruction of the material.

The Study of the Caudal Vertebrae of Thick-Toed Geckos after a Prolonged Space Flight by X-ray Phase-Contrast Micro-CT.

The proximal caudal vertebrae and notochord in thick-toed geckos (TG) (Chondrodactylus turneri, Gray, 1864) were investigated after a 30-day space flight onboard the biosatellite Bion-M1. This region has not been explored in previous studies. Our research focused on finding sites most affected by demineralization caused by microgravity (G0). We used X-ray phase-contrast tomography to study TG samples without invasive prior preparation to clarify our previous findings on the resistance of TG's bones to demineralization in G0. The results of the present study confirmed that geckos are capable of preserving bone mass after flight, as neither cortical nor trabecular bone volume fraction showed statistically significant changes after flight. On the other hand, we observed a clear decrease in the mineralization of the notochordal septum and a substantial rise in intercentrum volume following the flight. To monitor TG's mineral metabolism in G0, we propose to measure the volume of mineralized tissue in the notochordal septum. This technique holds promise as a sensitive approach to track the demineralization process in G0, given that the volume of calcification within the septum is limited, making it easy to detect even slight changes in mineral content.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

Deep Learning-Based Segmentation of Post-Mortem Human's Olfactory Bulb Structures in X-ray Phase-Contrast Tomography.

The human olfactory bulb (OB) has a laminar structure. The segregation of cell populations in the OB image poses a significant challenge because of indistinct boundaries of the layers. Standard 3D visualization tools usually have a low resolution and cannot provide the high accuracy required for morphometric analysis. X-ray phase contrast tomography (XPCT) offers sufficient resolution and contrast to identify single cells in large volumes of the brain. The numerous microanatomical structures detectable in XPCT image of the OB, however, greatly complicate the manual delineation of OB neuronal cell layers. To address the challenging problem of fully automated segmentation of XPCT images of human OB morphological layers, we propose a new pipeline for tomographic data processing. Convolutional neural networks (CNN) were used to segment XPCT image of native unstained human OB. Virtual segmentation of the whole OB and an accurate delineation of each layer in a healthy non-demented OB is mandatory as the first step for assessing OB morphological changes in smell impairment research. In this framework, we proposed an effective tool that could help to shed light on OB layer-specific degeneration in patients with olfactory disorder.

Resonance modes filtering in structured x-ray waveguides.

We discuss the self-imaging effect that occurs in a multimode planar x-ray waveguide (WG) with a nanometer vacuum gap, where an additional longitudinal periodicity has been imposed by a periodical structure (a micron scale step-like grating) on the reflecting sidewalls. Taking into account the general Montgomery conditions and the particular case of Talbot effect, we show that this additional longitudinal periodicity, if suitably designed, can filter out the asymmetric and the high order resonance modes, providing a coherent beam at the exit, even if the WG is illuminated by an incoherent source.

A computational platform for the virtual unfolding of Herculaneum Papyri.

Ancient Herculaneum papyrus scrolls, hopelessly charred in the 79 A.D. Vesuvius eruption, contain valuable writings of the Greek philosophers of the day, including works of the Epicurean Philodemus. X-ray phase contrast tomography has recently begun unlocking their secrets. However, only small portions of the text hidden inside the scroll have been recover. One of the challenging tasks in Herculaneum papyri investigation is their virtual unfolding because of their highly complicated structure and three-dimensional arrangement. Although this procedure is feasible, problems in segmentation and flattening hinder the unrolling of a large portion of papyrus. We propose a computational platform for the virtual unfolding procedure, and we show the results of its application on two Herculaneum papyrus fragments. This work paves the way to a comprehensive survey and to further interpretation of larger portions of text hidden inside the carbonized Herculaneum papyri.

Micro-CT Study of Mongolian Gerbil Humeral Bone After Prolonged Spaceflight Based on a New Algorithm for Delimitation of Long-Bone Regions.

The Mongolian gerbil displays unique physiological and anatomical features that make this species an attractive object for biological experiments in space. However, until recently, the Mongolian gerbil has remained a novel, mostly unstudied animal model in investigating bone loss in weightlessness (G0). After 12 days of orbital Foton-M3 mission, the humerus of Mongolian gerbils has been studied here via micro-computed tomography (micro-CT) to quantify bone morphometric parameters. The samples from the flight group, delayed synchronous ground-control group, and basal control group were investigated, and main morphometric parameters were reported in the article. The accurate selection of a region of interest is an essential step for a correct assessment of bone parameters. We proposed a new, easy and efficient method for delimiting the bone's basic regions in the humerus. It is based on quantitative estimation of X-ray attenuation in the cortical bone as a function of humerus bone length. The micro-CT analysis of the basic bone regions revealed a difference in bone morphometric parameters between the flight and control gerbils. The most significant bone loss was observed in the cortical part of the proximal humeral zone in the flight group. No statistically significant changes of volume fraction in the cancellous tissue of proximal and distal epiphyses and metaphyses were observed. A statistically significant increase in both cancellous bone volume and bone X-ray attenuation in the flight group was detected in the proximal part of the diaphyses. We assume that enhanced calcium deposition in the diaphyseal cancellous tissue occurred due to a bone response to G0 conditions.

Discovery of Lebambromyia in Myanmar Cretaceous Amber: Phylogenetic and Biogeographic Implications (Insecta, Diptera, Phoroidea).

Lebambromyia sacculifera sp. nov. is described from Late Cretaceous amber from Myanmar, integrating traditional observation techniques and X-ray phase contrast microtomography. Lebambromyia sacculifera is the second species of Lebambromyia after L. acrai Grimaldi and Cumming, described from Lebanese amber (Early Cretaceous), and the first record of this taxon from Myanmar amber, considerably extending the temporal and geographic range of this genus. The new specimen bears a previously undetected set of phylogenetically relevant characters such as a postpedicel sacculus and a prominent clypeus, which are shared with Ironomyiidae and Eumuscomorpha. Our cladistic analyses confirmed that Lebambromyia represented a distinct monophyletic lineage related to Platypezidae and Ironomyiidae, though its affinities are strongly influenced by the interpretation and coding of the enigmatic set of features characterizing these fossil flies.

3D Spatial Distribution of Nanoparticles in Mice Brain Metastases by X-ray Phase-Contrast Tomography.

Characterizing nanoparticles (NPs) distribution in multiple and complex metastases is of fundamental relevance for the development of radiological protocols based on NPs administration. In the literature, there have been advances in monitoring NPs in tissues. However, the lack of 3D information is still an issue. X-ray phase-contrast tomography (XPCT) is a 3D label-free, non-invasive and multi-scale approach allowing imaging anatomical details with high spatial and contrast resolutions. Here an XPCT qualitative study on NPs distribution in a mouse brain model of melanoma metastases injected with gadolinium-based NPs for theranostics is presented. For the first time, XPCT images show the NPs uptake at micrometer resolution over the full brain. Our results revealed a heterogeneous distribution of the NPs inside the melanoma metastases, bridging the gap in spatial resolution between magnetic resonance imaging and histology. Our findings demonstrated that XPCT is a reliable technique for NPs detection and can be considered as an emerging method for the study of NPs distribution in organs.

Corrigendum: X-ray Phase Contrast Tomography Serves Preclinical Investigation of Neurodegenerative Diseases.

[This corrects the article DOI: 10.3389/fnins.2020.584161.].

Analysis of tapered front-coupling X-ray waveguides.

The coupling and propagation of electromagnetic waves through planar X-ray waveguides (WG) with vacuum gap and Si claddings are analyzed in detail, starting from the source and ending at the detector. The general case of linearly tapered WGs (i.e. with the entrance aperture different from the exit one) is considered. Different kinds of sources, i.e. synchrotron radiation and laboratory desk-top sources, have been considered, with the former providing a fully coherent incoming beam and the latter partially coherent beams. It is demonstrated that useful information about the parameters of the WG can be derived, comparing experimental results with computer simulation based on analytical solutions of the Helmholtz equation which take into account the amplitude and phase matching between the standing waves created in front of the WG, and the resonance modes propagating into the WG.

X-ray phase contrast microscopy at 300 nm resolution with laboratory sources.

We report the performance of an X-ray phase contrast microscope for laboratory sources with 300 nm spatial resolution. The microscope is based on a commercial X-ray microfocus source equipped with a planar X-ray waveguide able to produce a sub-micrometer x-ray beam in one dimension. Phase contrast images of representative samples are reported. The achieved contrast and resolution is discussed for different configurations. The proposed approach could represent a simple, inexpensive, solution for sub-micrometer resolution imaging with small laboratory setups.

Assessment of plaque morphology in Alzheimer's mouse cerebellum using three-dimensional X-ray phase-based virtual histology.

Visualization and characterization of [Formula: see text]-amyloid deposits is a fundamental task in pre-clinical study of Alzheimer's disease (AD) to assess its evolution and monitor the efficiency of new therapeutic strategies. While the cerebellum is one of the brain areas most underestimated in the context of AD, renewed interest in cerebellar lesions has recently arisen as they may link to motor and cognitive alterations. Thus, we quantitatively investigated three-dimensional plaque morphology in the cerebellum in APP/PS1 transgenic mouse, as a model of AD. In order to obtain a complete high-resolution three-dimensional view of the investigated tissue, we exploited synchrotron X-ray phase contrast tomography (XPCT), providing virtual slices with histology-matching resolution. We found the formation of plaques elongated in shape, and with a specific orientation in space depending on the investigated region of the cerebellar cortex. Remarkably, a similar shape is observed in human cerebellum from demented patients. Our findings demonstrate the capability of XPCT in volumetric quantification, supporting the current knowledge about plaque morphology in the cerebellum and the fundamental role of the surrounding tissue in driving their evolution. A good correlation with the human neuropathology is also reported.