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Abstract Detecting breast tissue alterations is essential for cancer diagnosis. However, inherent bidimensionality limits histological procedures’ effectiveness in identifying these changes. Our study applies a 3D virtual histology method based on X-ray phase-contrast microtomography (PhC $$\mu$$ μ CT), performed at a synchrotron facility, to investigate breast tissue samples including different types of lesions, namely intraductal papilloma, micropapillary intracystic carcinoma, and invasive lobular carcinoma. One-to-one comparisons of X-ray and histological images explore the clinical potential of 3D X-ray virtual histology. Results show that PhC $$\mu$$ μ CT technique provides high spatial resolution and soft tissue sensitivity, while being non-destructive, not requiring a dedicated sample processing and being compatible with conventional histology. PhC $$\mu$$ μ CT can enhance the visualization of morphological characteristics such as stromal tissue, fibrovascular core, terminal duct lobular unit, stromal/epithelium interface, basement membrane, and adipocytes. Despite not reaching the (sub) cellular level, the three-dimensionality of PhC $$\mu$$ μ CT images allows to depict in-depth alterations of the breast tissues, potentially revealing pathologically relevant details missed by a single histological section. Compared to serial sectioning, PhC $$\mu$$ μ CT allows the virtual investigation of the sample volume along any orientation, possibly guiding the pathologist in the choice of the most suitable cutting plane. Overall, PhC $$\mu$$ μ CT virtual histology holds great promise as a tool adding to conventional histology for improving efficiency, accessibility, and diagnostic accuracy of pathological evaluation.

Synchrotron radiation propagation-based phase-contrast micro-computed tomography (SRμCT) has been used increasingly for detailed characterization of tissue specimens, alternative to classical histology. It allows rapid and non-destructive three-dimensional (3D) virtual histology of unstained specimens. Although many studies show that standard protocols for formalin-fixation and paraffin-embedded (FFPE) tissue specimens are well suited for propagation based phase-contrast imaging (PBI), refinement of sample preparation protocols for this relatively recent development is still in its early stage. There are several limitations using FFPE blocks including air-bubble inclusion, and crack formation, which affect the quality of the micro-CT scans. In this study we demonstrate that optical clearing of colon cancer specimen followed by embedding in Phytagel, as an alternative sample preparation protocol yields comparable PBI micro-CT image quality and mitigates the aforementioned drawbacks of FFPE specimens. Moreover, since single-distance phase retrieval algorithms for PBI image reconstruction expect scanned materials to be either weakly-absorbing or having a fixed ratio between absorption and phase shift, we hypothesize that optical clearing will result in specimens that are well in line with these mathematical assumption of the applied phase retrieval algorithm (homogeneous form of the Transport-of-Intensity equation TIE-HOM). In addition, we show that classical histological analysis is still possible after re-embedding of the specimen in paraffin. Thus, the proposed novel workflow to scan optical cleared specimens using SRμCT can be used in combination with light sheet microscopy and can be integrated in a classical pipeline for pathological tissue characterization.

IntroductionSynchrotron-based propagation-based imaging (PBI) is ideally suited for lung imaging and has successfully been applied in a variety of in vivo small animal studies. Virtually all these experiments were tailored to achieve extremely high spatial resolution close to the alveolar level while delivering high x-ray doses that would not permit longitudinal studies. However, the main rationale for performing lung imaging studies in vivo in small animal models is the ability to follow disease progression or monitor treatment response in the same animal over time. Thus, an in vivo imaging strategy should ideally allow performing longitudinal studies.MethodsHere, we demonstrate our findings of using PBI-based planar and CT imaging with two different detectors—MÖNCH 0.3 direct conversion detector and a complementary metal-oxide-semiconductor (CMOS) detector (Photonics Science)—in an Ovalbumin induced experimental allergic airway disease mouse model in comparison with healthy controls. The mice were imaged free breathing under isoflurane anesthesia.ResultsAt x-ray dose levels below those once used by commercial small animal CT devices at similar spatial resolutions, we were able to resolve structural changes at a pixel size down to 25 μm and demonstrate the reduction in elastic recoil in the asthmatic mice in cinematic planar x-ray imaging with a frame rate of up to 100 fps.DiscussionThus, we believe that our approach will permit longitudinal small animal lung disease studies, closely following the mice over longer time spans.

X-ray phase-contrast micro computed tomography using synchrotron radiation (SR PhC-µCT) offers unique 3D imaging capabilities for visualizing microstructure of the human brain. Its applicability for unstained soft tissue is an area of active research. Acquiring images from a tissue block without needing to section it into thin slices, as required in routine histology, allows for investigating the microstructure in its natural 3D space. This paper presents a detailed step-by-step guideline for imaging unstained human brain tissue at resolutions of a few micrometers with SR PhC-µCT implemented at SYRMEP, the hard X-ray imaging beamline of Elettra, the Italian synchrotron facility. We present examples of how blood vessels and neurons appear in the images acquired with isotropic 5 μm and 1 µm voxel sizes. Furthermore, the proposed protocol can be used to investigate important biological substrates such as neuromelanin or corpora amylacea. Their spatial distribution can be studied using specifically tailored segmentation tools that are validated by classical histology methods. In conclusion, SR PhC-µCT using the proposed protocols, including data acquisition and image processing, offers viable means of obtaining information about the anatomy of the human brain at the cellular level in 3D.

This work introduces a novel setup for computed tomography of heavy and bulky specimens at the SYRMEP beamline of the Italian synchrotron Elettra. All the key features of the setup are described and the first application to off-center computed tomography scanning of a human chest phantom (approximately 45 kg) as well as the first results for vertical helical acquisitions are discussed.

Abstract Absorption-based clinical computed tomography (CT) is the current imaging method of choice in the diagnosis of lung diseases. Many pulmonary diseases are affecting microscopic structures of the lung, such as terminal bronchi, alveolar spaces, sublobular blood vessels or the pulmonary interstitial tissue. As spatial resolution in CT is limited by the clinically acceptable applied X-ray dose, a comprehensive diagnosis of conditions such as interstitial lung disease, idiopathic pulmonary fibrosis or the characterization of small pulmonary nodules is limited and may require additional validation by invasive lung biopsies. Propagation-based imaging (PBI) is a phase sensitive X-ray imaging technique capable of reaching high spatial resolutions at relatively low applied radiation dose levels. In this publication, we present technical refinements of PBI for the characterization of different artificial lung pathologies, mimicking clinically relevant patterns in ventilated fresh porcine lungs in a human-scale chest phantom. The combination of a very large propagation distance of 10.7 m and a photon counting detector with $$100\,\upmu \hbox {m}$$ 100 μ m pixel size enabled high resolution PBI CT with significantly improved dose efficiency, measured by thermoluminescence detectors. Image quality was directly compared with state-of-the-art clinical CT. PBI with increased propagation distance was found to provide improved image quality at the same or even lower X-ray dose levels than clinical CT. By combining PBI with iodine k-edge subtraction imaging we further demonstrate that, the high quality of the calculated iodine concentration maps might be a potential tool for the analysis of lung perfusion in great detail. Our results indicate PBI to be of great value for accurate diagnosis of lung disease in patients as it allows to depict pathological lesions non-invasively at high resolution in 3D. This will especially benefit patients at high risk of complications from invasive lung biopsies such as in the setting of suspected idiopathic pulmonary fibrosis (IPF).

Precise morphological analysis of tumour tissue samples is crucial for accurate diagnosis and staging of colorectal cancer (CRC), but remains limited by the 2D nature of conventional histology. Our aim is to offer a 3D representation of tissue samples by means of X-ray-based imaging to facilitate the evaluation of clinically relevant features in cancer tissue, a process that is currently subject to various restrictions. In this study, we show that propagation-based synchrotron radiation-based free propagation phase-contrast microcomputed tomography (SRµCT) is suitable for the generation of 3D tumour volumes with 2-µm voxel size using standard formalin-fixed, paraffin-embedded tissue from CRC patients and provides sufficient contrast for virtual histology. We demonstrate that, using an existing registration pipeline, a 2D histologic haematoxylin–eosin slice can be placed in the context of the 3D µCT volume. The precisely registered histologic section can then be used as a “seed point” for the segmentation and depiction of major histologic features. This approach allows for a more comprehensive understanding of the organisation of the tumour in space with respect to other structures such as vessels, fat, and lymph nodes, and has the potential to improve patients’ prognostic outcomes.

Corpora amylacea (CA) are polyglucosan aggregated granules that accumulate in the human body throughout aging. In the cerebrum, CA have been found in proximity to ventricular walls, pial surfaces, and blood vessels. However, studies showing their three-dimensional spatial distribution are sparse. In this study, volumetric images of four human brain stems were obtained with MRI and phase-contrast X-ray microtomography, followed up by Periodic acid Schiff stain for validation. CA appeared as hyperintense spheroid structures with diameters up to 30 μm. An automatic pipeline was developed to segment the CA, and the spatial distribution of over 200,000 individual corpora amylacea could be investigated. A threefold—or higher—density of CA was detected in the dorsomedial column of the periaqueductal gray (860–4,200 CA count/mm3) than in the superior colliculus (150–340 CA count/mm3). We estimated that about 2% of the CA were located in the immediate vicinity of the vessels or in the peri-vascular space. While CA in the ependymal lining of the cerebral aqueduct was rare, the sub-pial tissue of the anterior and posterior midbrain contained several CA. In the sample with the highest CA density, quantitative maps obtained with MRI revealed high R2* values and a diamagnetic shift in a region which spatially coincided with the CA dense region.

X-ray phase-contrast tomography offers a highly sensitive 3D imaging approach to investigate different disease-relevant networks at levels ranging from single cell through to intact organ. The authors present a concomitant study of the evolution of tissue damage and inflammation in different organs affected by the disease in the murine model for multiple sclerosis.

While advanced imaging strategies have improved the diagnosis of bone-related pathologies, early signs of bone alterations remain difficult to detect. The Covid-19 pandemic has brought attention to the need for a better understanding of bone micro-scale toughening and weakening phenomena. This study used an artificial intelligence-based tool to automatically investigate and validate four clinical hypotheses by examining osteocyte lacunae on a large scale with synchrotron image-guided failure assessment. The findings indicate that trabecular bone features exhibit intrinsic variability related to external loading, micro-scale bone characteristics affect fracture initiation and propagation, osteoporosis signs can be detected at the micro-scale through changes in osteocyte lacunar features, and Covid-19 worsens micro-scale porosities in a statistically significant manner similar to the osteoporotic condition. Incorporating these findings with existing clinical and diagnostic tools could prevent micro-scale damages from progressing into critical fractures.

Abstract Characterizing the microvasculature of the human brain is critical to advance understanding of brain vascular function. Most methods rely on tissue staining and microscopy in two-dimensions, which pose several challenges to visualize the three-dimensional structure of microvessels. In this study, we used an edge-based segmentation method to extract the 3D vasculature from synchrotron radiation phase-contrast microtomography (PC-μCT) of two unstained, paraffin-embedded midbrain region of the human brain stem. Vascular structures identified in PC-μCT were validated with histology of the same specimen. Using the Deriche-Canny edge detector that was sensitive to the boundary between tissue and vascular space, we could segment the vessels independent of signal variations in PC-μCT images. From the segmented volumetric vasculature, we calculated vessel diameter, vessel length and volume fraction of the vasculature in the superior colliculi. From high resolution images, we found the most frequent vessel diameter to be between 8.6–10.2 µm. Our findings are consistent with the known anatomy showing two types of vessels with distinctive morphology: peripheral collicular vessels and central collicular vessels. The proposed method opens up new possibilities for vascular research of the central nervous system using synchrotron radiation PC-μCT of unstained human tissue.

Background: Histopathological analysis represents the gold standard in clinical practice for diagnosing skin neoplasms. While the current diagnostic workflow has specialized in producing robust and accurate results, interpreting tissue architecture and malignant cellular morphology correctly remains one of the greatest challenges for pathologists. This paper aims to explore the prospect of applying x‐ray virtual histology to human skin tumor excisions and correlating it with the histological validation. Materials and Methods: Seven skin biopsies containing intriguing melanoma types and pigmented skin lesions were scanned using x‐ray Computed micro‐Tomography (μCT) and then sectioned for conventional histology assessment. Results: The tissue microarchitecture reconstructed by μCT offers detailed insights into diagnosing the malignancy or benignity of the skin lesions. Three‐dimensional reconstruction via x‐ray virtual histology reveals infiltrative patterns in basal cell carcinoma and evaluated invasiveness in melanoma. The technology enables the identification of pagetoid distributions of neoplastic cells and the assessment of melanoma depth in three dimensions. Conclusion: Although the proposed approach is not intended to replace conventional histology, the non‐destructive nature of the sample and the clarity provided by virtual inspection demonstrate the promising impact of μCT as a valid support method prior to conventional histological sectioning. Indeed, μCT images can suggest the optimal sectioning position before using a microtome, as is commonly performed in histological practice. Moreover, the three‐dimensional nature of the proposed approach paves the way for a more accurate assessment of significant prognostic factors in melanoma, such as Breslow thickness, by considering the whole micro‐volume rather than a two‐dimensional observation. [ABSTRACT FROM AUTHOR]

Synchrotron radiation propagation-based phase-contrast micro-computed tomography (SRμCT) has been used increasingly for detailed characterization of tissue specimens, alternative to classical histology. It allows rapid and nondestructive three-dimensional (3D) virtual histology of unstained specimens. Although many studies show that standard protocols for formalin-fixation and paraffin-embedded (FFPE) tissue specimens are well suited for propagation based phase-contrast imaging (PBI), refinement of sample preparation protocols for this relatively recent development is still in its early stage. There are several limitations using FFPE blocks including air-bubble inclusion, and crack formation, which affect the quality of the micro-CT scans. In this study we demonstrate that optical clearing of colon cancer specimen followed by embedding in Phytagel, as an alternative sample preparation protocol yields comparable PBI micro-CT image quality and mitigates the aforementioned drawbacks of FFPE specimens. Moreover, since single-distance phase retrieval algorithms for PBI image reconstruction expect scanned materials to be either weakly-absorbing or having a fixed ratio between absorption and phase shift, we hypothesize that optical clearing will result in specimens that are well in line with these mathematical assumption of the applied phase retrieval algorithm (homogeneous form of the Transport-of-Intensity equation TIE-HOM). In addition, we show that classical histological analysis is still possible after re-embedding of the specimen in paraffin. Thus, the proposed novel workflow to scan optical cleared specimens using SRμCT can be used in combination with light sheet microscopy and can be integrated in a classical pipeline for pathological tissue characterization. [ABSTRACT FROM AUTHOR]

Objective: Early diagnosis of syndromic monogenic diabetes allows for proper management and can lead to improved quality of life in the long term. This report aimed to describe 2 genetically confirmed cases of Wolfram syndrome, a rare endoplasmic reticulum disorder characterized by insulin-dependent diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Case Report: A 16-year-old Caucasian male patient and a 25-year-old Caucasian female patient with a history of diabetes mellitus and optic nerve atrophy presented at our medical center. Both patients were initially diagnosed with type 1 diabetes but negative for islet autoantibodies. Their body mass indexes were under 25 at the diagnosis. Their history and presentation were highly suspicious for Wolfram syndrome. Discussion: The genetic tests revealed a known Wolfram syndrome 1 (WFS1) pathogenic variant (homozygous) in the 16-year-old male patient and 2 known WFS1 pathogenic variants (compound heterozygous) in the 25-year-old female patient with diabetes mellitus and optic nerve atrophy, confirming the diagnosis of Wolfram syndrome. The first patient had a moderate form, and the second patient had a milder form of Wolfram syndrome. Conclusion: Providers should consider monogenic diabetes genetic testing, including WFS1 gene, for patients with early-onset diabetes who are negative for islet autoantibodies and lean. Two patients described in this article could have been diagnosed with Wolfram syndrome before they developed optic nerve atrophy. Genetic testing is a valuable tool for the early detection of Wolfram syndrome, which leads to proper management and improved quality of life in patients with this rare medical condition.

Abstract Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.

The prevalence of postpartum thyroiditis (PPT) averages 5%, with a range from 1% (Thailand) to 22% (Wales, UK, and Liguria, Italy), but 3.6% in another Italian region (Puglia). Evolution of PPT into permanent hypothyroidism (PH) occurs in approximately 50% of cases. Positive thyroperoxidase autoantibodies (TPOAb) in a pregnant woman is a strong predictor of PPT. Because in previous gestational cohorts we found an approximate 12% rate of TPOAb positivity, which compares with 15% in the Liguria cohort and 6% in the Puglia cohort, we hypothesized that the currently unknown prevalence of PPT in Sicily would approximate the said Liguria prevalence. We also explored the predictive value of serum thyroglobulin Ab (TgAb) positivity and ultrasonographic signs suggestive of thyroiditis (UST) at first trimester of gestation for PPT.Of 412 pregnant women who were followed-up for 1 year after delivery, 63 (15.3%) developed PPT, and 54% of them had PH. Gestational rates of TPOAb positivity alone, TgAb positivity alone or UST were 11.4%, 7.8% or 35.0%, with associated PPT rates of 66%, 45% or 36%. TgAb assay allowed detection of 9/63 PPT women (14.3%) who were TPOAb-negative. However, TPOAb remained a better predictor compared to TgAb or UST (odds ratio = 32 vs 10 or 13). Lowering the positivity threshold for either Ab to ≥61 U/ml, Ab-positive were 23.8% of PPT women and 17.7% of pH women. UST was detected in 82.5% of women who developed PPT, precisely 88% of those who evolved into PH and 75.9% of those who did not.Ours is the second study of the new millennium showing a PPT frequency >10%. The dual Ab and lowered threshold strategy correctly predicts more cases of PPT and PH compared to the sole TPOAb strategy. We confirm that half of the PPT women will have PH. Keywords: Geoepidemiology, Postpartum thyroiditis, Thyroid autoimmunity