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Background: Mitochondria are highly dynamic organelles that constantly undergo processes of fission and fusion. The changes in mitochondrial dynamics shape the organellar morphology and influence cellular activity regulation. Soft X-ray tomography (SXT) allows for three-dimensional imaging of cellular structures while they remain in their natural, hydrated state, which omits the need for cell fixation and sectioning. Synchrotron facilities globally primarily use flat grids as sample carriers for SXT analysis, focusing on adherent cells. To investigate mitochondrial morphology and structure in hydrated yeast cells using SXT, it is necessary to establish a method that employs the flat grid system for examining cells in suspension., Results: We developed a procedure to adhere suspended yeast cells to a flat grid for SXT analysis. Using this protocol, we obtained images of wild-type yeast cells, strains with mitochondrial dynamics defects, and mutant cells possessing distinctive mitochondria. The SXT images align well with the results from fluorescent microscopy. Optimized organellar visualization was achieved by constructing three-dimensional models of entire yeast cells., Conclusions: In this study, we characterized the mitochondrial network in yeast cells using SXT. The optimized sample preparation procedure was effective for suspended cells like yeast, utilizing a flat grid system to analyze mitochondrial structure through SXT. The findings corresponded with the mitochondrial morphology observed under fluorescence microscopy, both in regular and disrupted dynamic equilibrium. With the acquired image of unique mitochondria in Δhap2 cells, our results revealed that intricate details of organelles, such as mitochondria and vacuoles in yeast cells, can be characterized using SXT. Therefore, this optimized system supports the expanded application of SXT for studying organellar structure and morphology in suspended cells., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Heterochromatin organization is critical to many genome-related programs including transcriptional silencing and DNA repair. While super-resolution imaging, electron microscopy, and multiomics methods have provided indirect insights into the heterochromatin organization, a direct measurement of mesoscale heterochromatin ultrastructure is still missing. We use a combination of correlative light microscopy and cryo-soft X-ray tomography (CLXT) to analyze heterochromatin organization in the intact hydrated state of human mammary fibroblast cells. Our analysis reveals that the heterochromatin ultra-structure has a typical mean domain size of approximately 80 nm and a mean separation of approximately 120 nm between domains. Functional perturbations yield further insights into the molecular density and alterations in the mesoscale organization of the heterochromatin regions. Furthermore, our polymer simulations provide a mechanistic basis for the experimentally observed size and separation distributions of the mesoscale chromatin domains. Collectively, our results provide direct, label-free observation of heterochromatin organization in the intact hydrated state of cells., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

A new species of Helicascus satunensis sp. nov. was collected on mature dead fruits of the Nypa palm in Satun Province, southern Thailand. Its morphological characteristics are similar to those of the genus Helicascus . Recently, a genus Helicascus with three species from marine habitats worldwide was studied. The morphology of this fungus was investigated and combined with multigene sequence analyzes of small subunit (SSU), large subunit (LSU), internal transcribed spacer (ITS) ribosomal DNA, translation elongation factor 1-alpha (TEF-1 α ) and RNA polymerase II (RPB2) genes. Morphologically, H. satunensis sp. nov. is characterized by semi-immersed, lenticular ascomata, multilocules, a bitunicate ascus and smooth, obovoid, dark brown ascospores that are one-septate and unequally two-celled. In addition, 3D visualization using synchrotron X-ray tomography was performed to investigate the interaction between fruiting body and substrata. Molecular phylogeny with multigene revealed that H. satunensis sp. nov. belongs to the family Morosphaeriaceae, order Pleosporales, class Dothideomycetes. Furthermore, H. satunensis sp. nov. forms a well-supported clade with Helicascus species described from marine habitats. Based on the unique morphological and molecular evidence, we propose this fungus, H. satunensis sp. nov., as a new species for Helicascus ., Competing Interests: The authors declare there are no competing interests., (©2024 Preedanon et al.)

Three-dimensional correlative multimodal and multiscale imaging is an emerging method for investigating the complex hierarchical structure of biological materials such as bone. This approach synthesizes images acquired across multiple length scales, for the same region of interest, to provide a comprehensive view of the material structure of a sample. Here, we develop a workflow for the structural analysis of human trabecular bone using a femtosecond laser to produce a precise grid to facilitate correlation between imaging modalities and identification of structures of interest, in this case, a single trabecula within a volume of trabecular bone. Through such image registration, high resolution X-ray microscopy imaging revealed fine architectural details, including the cement sheath and bone cell lacunae of the selected bone trabecula. The selected bone volume was exposed with a combination of manual polishing and site-specific femtosecond laser ablation and then examined with plasma focused ion beam-scanning electron microscopy. This reliable and versatile correlation approach has the potential to be applied to a variety of biological tissues and traditional engineered materials. The proposed workflow has the enhanced capability for generating highly resolved and broadly contextualized structural data for a better understanding of the architectural features of a material spanning its macroscopic to nanoscopic levels., (© 2024. The Author(s).)

Correlative imaging of fluorescence microscopy and soft X-ray microscopy plays a crucial role in exploring the relationship between structure and function in cellular biology. However, the current correlative imaging methods are limited either to off-line or low-resolution fluorescence imaging. In this study, we developed an integrated on-line cryogenic photoactivated localization microscopy (cryo-PALM) system at a soft X-ray microscopy station. This design eliminates some critical issues such as sample damage and complex post-correlation arising from transferring samples between different cryostages. Furthermore, we successfully achieved correlative imaging of cryopreserved near-native cells, with a resolution of about 50 nm of cryo-PALM. Therefore, the developed on-line correlation imaging platform provides a powerful tool for investigating the intricate relationship between structure and function in biological and molecular interactions, as well as in other life science disciplines.

The dysfunction of α and β cells in pancreatic islets can lead to diabetes. Many questions remain on the subcellular organization of islet cells during the progression of disease. Existing three-dimensional cellular mapping approaches face challenges such as time-intensive sample sectioning and subjective cellular identification. To address these challenges, we have developed a subcellular feature-based classification approach, which allows us to identify α and β cells and quantify their subcellular structural characteristics using soft X-ray tomography (SXT). We observed significant differences in whole-cell morphological and organelle statistics between the two cell types. Additionally, we characterize subtle biophysical differences between individual insulin and glucagon vesicles by analyzing vesicle size and molecular density distributions, which were not previously possible using other methods. These sub-vesicular parameters enable us to predict cell types systematically using supervised machine learning. We also visualize distinct vesicle and cell subtypes using Uniform Manifold Approximation and Projection (UMAP) embeddings, which provides us with an innovative approach to explore structural heterogeneity in islet cells. This methodology presents an innovative approach for tracking biologically meaningful heterogeneity in cells that can be applied to any cellular system.

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer's disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

During starvation in the yeast Saccharomyces cerevisiae vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep-learning computational approach to visualize and quantify these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 ( ncr1∆ cells) or Npc2 ( npc2∆ cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that ncr1∆ and npc2∆ cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells. Abbreviations: BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza- s -Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; npc2∆ , yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, N -(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; ncr1∆ , yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD 600 , optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.

Cryo-soft X-ray tomography is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The sample preparation is relatively straightforward; requiring just fast freezing of the specimen before transfer to the microscope for imaging. It is also possible to image chemically fixed samples where necessary. The technique can be correlated with cryo fluorescence microscopy to localize fluorescent proteins to organelles within the whole cell volume. Cryo-correlated light and soft X-ray tomography is particularly useful for the study of gross morphological changes brought about by disease or drugs. For example, viral fluorescent tags can be co-localized to sites of viral replication in the soft X-ray volume. In general this approach is extremely useful in the study of complex 3D organelle structure, nanoparticle uptake or in the detection of rare events in the context of whole cell structure. The main challenge of soft X-ray tomography is that the soft X-ray illumination required for imaging has heretofore only been available at a small number of synchrotron labs worldwide. Recently, a compact device with a footprint small enough to fit in a standard laboratory setting has been deployed ("the SXT-100") and is routinely imaging cryo prepared samples addressing a variety of disease and drug research applications. The SXT-100 facilitates greater access to this powerful technique and greatly increases the scope and throughput of potential research projects. Furthermore, the availability of cryo-soft X-ray tomography in the laboratory will accelerate the development of novel correlative and multimodal workflows by integration with light and electron microscope based approaches. It also allows for co-location of this powerful imaging modality at BSL3 labs or other facilities where safety or intellectual property considerations are paramount. Here we describe the compact SXT-100 microscope along with its novel integrated cryo-fluorescence imaging capability., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.

Soft X-ray tomography (SXT) is an imaging technique to visualise whole cells without fixation, staining, and sectioning. For SXT imaging, cells are cryopreserved and imaged at cryogenic conditions. Such 'near-to-native' state imaging is in high demand and initiated the development of the laboratory table-top SXT microscope. As many laboratories do not have access to cryogenic equipment, we asked ourselves whether SXT imaging is feasible on dry specimens. This paper shows how the dehydration of cells can be used as an alternative sample preparation to obtain ultrastructure information. We compare different dehydration processes on mouse embryonic fibroblasts in terms of ultrastructural preservation and shrinkage. Based on this analysis, we chose critical point (CPD) dried cells for SXT imaging. In comparison to cryopreserved and air-dried cells, CPD dehydrated cells show high structural integrity although with about 3-7 times higher X-ray absorption for cellular organelles. As the difference in X-ray absorption values between organelles is preserved, 3D anatomy of CPD-dried cells can be segmented and analysed, demonstrating the applicability of CPD-dried sample preparation for SXT imaging. LAY DESCRIPTION: Soft X-ray tomography (SXT) is an imaging technique that allows to see the internal structures of cells without the need for special treatments like fixation or staining. Typically, SXT imaging involves freezing and imaging cells at very low temperatures. However, since many labs lack the necessary equipment, we explored whether SXT imaging could be done on dry samples instead. We compared different dehydration methods and found that critical point drying (CPD) was the most promising for SXT imaging. CPD-dried cells showed high structural integrity, although they absorbed more X-rays than hydrated cells, demonstrating that CPD-dried sample preparation is a viable alternative for SXT imaging., (© 2023 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Cryo-soft X-ray tomography (cryo-SXT) is a powerful method to investigate the ultrastructure of cells, offering resolution in the tens of nanometer range and strong contrast for membranous structures without requiring labeling or chemical fixation. The short acquisition time and the relatively large field of view leads to fast acquisition of large amounts of tomographic image data. Segmentation of these data into accessible features is a necessary step in gaining biologically relevant information from cryo-soft X-ray tomograms. However, manual image segmentation still requires several orders of magnitude more time than data acquisition. To address this challenge, we have here developed an end-to-end automated 3D segmentation pipeline based on semisupervised deep learning. Our approach is suitable for high-throughput analysis of large amounts of tomographic data, while being robust when faced with limited manual annotations and variations in the tomographic conditions. We validate our approach by extracting three-dimensional information on cellular ultrastructure and by quantifying nanoscopic morphological parameters of filopodia in mammalian cells.

Rational and Objectives: Comparison of radiation dose and image quality in routine abdominal and pelvic contrast-enhanced computed tomography (CECT) between a photon-counting detector CT (PCD-CT) and a dual energy dual source CT (DSCT)., Materials and Methods: 70 oncologic patients (mean age 66 ± 12 years, 29 females) were prospectively enrolled between November 2021 and February 2022. Abdominal CECT were clinically indicated and performed first on a 2 nd -generation DSCT and at follow-up on a 1 st -generation dual-source PCD-CT. The same contrast media (Imeron 350, Bracco imaging) and pump protocol was used for both scans. For both scanners, polychromatic images were reconstructed with 3mm slice thickness and comparable kernel (I30f[DSCT] and Br40f[PCD-CT]); for PCD-CT data from all counted events above the lowest energy threshold at 20 keV ("T3D") were used. Results were compared in terms of radiation dose metrics of CT dose index (CTDI vol ), dose length product (DLP) and size-specific dose estimation (SSDE), objective and subjective measurements of image quality were scored by two emergency radiologists including lesion conspicuity., Results: Median time interval between the scans was 4 months (IQR: 3-6). CNR vessel and SNR vessel of T3D reconstructions from PCD-CT were significantly higher than those of DSCT (all, p < 0.05). Qualitative image noise analysis from PCD-CT and DSCT yielded a mean of 4 each. Lesion conspicuity was rated significantly higher in PCD-CT (Q3 strength) compared to DSCT images. CTDI, DLP and SSDE mean values for PCD-CT and DSCT were 7.98 ± 2.56 mGy vs. 14.11 ± 2.92 mGy, 393.13 ± 153.55 mGy*cm vs. 693.61 ± 185.76 mGy*cm and 9.98 ± 2.41 vs. 14.63 ± 1.63, respectively, translating to a dose reduction of around 32% (SSDE)., Conclusion: PCD-CT enables oncologic abdominal CT with a significantly reduced dose while keeping image quality similar to 2 nd -generation DSCT., (Copyright © 2022 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Viruses are obligate intracellular pathogens that depend on their host cell machinery and metabolism for their replicative life cycle. Virus entry, replication, and assembly are dynamic processes that lead to the reorganisation of host cell components. Therefore, a complete understanding of the viral processes requires their study in the cellular context where advanced imaging has been proven valuable in providing the necessary information. Among the available imaging techniques, soft X-ray tomography (SXT) at cryogenic temperatures can provide three-dimensional mapping to 25 nm resolution and is ideally suited to visualise the internal organisation of virus-infected cells. In this chapter, the principles and practices of synchrotron-based cryo-soft X-ray tomography (cryo-SXT) in virus research are presented. The potential of the cryo-SXT in correlative microscopy platforms is also demonstrated through working examples of reovirus and hepatitis research at Beamline B24 (Diamond Light Source Synchrotron, UK) and BL09-Mistral beamline (ALBA Synchrotron, Spain), respectively., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Developing in silico models that accurately reflect a whole, functional cell is an ongoing challenge in biology. Current efforts bring together mathematical models, probabilistic models, visual representations, and data to create a multi-scale description of cellular processes. A realistic whole-cell model requires imaging data since it provides spatial constraints and other critical cellular characteristics that are still impossible to obtain by calculation alone. This review introduces Soft X-ray Tomography (SXT) as a powerful imaging technique to visualize and quantify the mesoscopic (~25 nm spatial scale) organelle landscape in whole cells. SXT generates three-dimensional reconstructions of cellular ultrastructure and provides a measured structural framework for whole-cell modeling. Combining SXT with data from disparate technologies at varying spatial resolutions provides further biochemical details and constraints for modeling cellular mechanisms. We conclude, based on the results discussed here, that SXT provides a foundational dataset for a broad spectrum of whole-cell modeling experiments., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Upon infection, viruses hijack the cell machinery and remodel host cell structures to utilize them for viral proliferation. Since viruses are about a thousand times smaller than their host cells, imaging virus-host interactions at high spatial resolution is like looking for a needle in a haystack. Scouting gross cellular changes with fluorescent microscopy is only possible for well-established viruses, where fluorescent tagging is developed. Soft X-ray tomography (SXT) offers 3D imaging of entire cells without the need for chemical fixation or labeling. Here, we use full-rotation SXT to visualize entire human B cells infected by the herpes simplex virus 1 (HSV-1). We have mapped the temporospatial remodeling of cells during the infection and observed changes in cellular structures, such as the presence of cytoplasmic stress granules and multivesicular structures, formation of nuclear virus-induced dense bodies, and aggregates of capsids. Our results demonstrate the power of SXT imaging for scouting virus-induced changes in infected cells and understanding the orchestration of virus-host remodeling quantitatively.

In the world of bioimaging, every choice made determines the quality and content of the data collected. The choice of imaging techniques for a study could showcase or dampen expected outcomes. Synchrotron radiation is indispensable for biomedical research, driven by the need to see into biological materials and capture intricate biochemical and biophysical details at controlled environments. The same need drives correlative approaches that enable the capture of heterologous but complementary information when studying any one single target subject. Recently, the applicability of one such synchrotron technique in bioimaging, soft X-ray tomography (SXT), facilitates exploratory and basic research and is actively progressing towards filling medical and industrial needs for the rapid screening of biomaterials, reagents and processes of immediate medical significance. Soft X-ray tomography at cryogenic temperatures (cryoSXT) fills the imaging resolution gap between fluorescence microscopy (in the hundreds of nanometers but relatively accessible) and electron microscopy (few nanometers but requires extensive effort and can be difficult to access). CryoSXT currently is accessible, fully documented, can deliver 3D imaging to 25 nm resolution in a high throughput fashion, does not require laborious sample preparation procedures and can be correlated with other imaging techniques. Here, we present the current state of SXT and outline its place within the bioimaging world alongside a guided matrix that aids decision making with regards to the applicability of any given imaging technique to a particular project. Case studies where cryoSXT has facilitated a better understanding of biological processes are highlighted and future directions are discussed., (© 2022 The Author(s).)

Inter-organelle interactions are a vital part of normal cellular function; however, these have proven difficult to quantify due to the range of scales encountered in cell biology and the throughput limitations of traditional imaging approaches. Here, we demonstrate that soft X-ray tomography (SXT) can be used to rapidly map ultrastructural reorganization and inter-organelle interactions in intact cells. SXT takes advantage of the naturally occurring, differential X-ray absorption of the carbon-rich compounds in each organelle. Specifically, we use SXT to map the spatiotemporal evolution of insulin vesicles and their co-localization and interaction with mitochondria in pancreatic β cells during insulin secretion and in response to different stimuli. We quantify changes in the morphology, biochemical composition, and relative position of mitochondria and insulin vesicles. These findings highlight the importance of a comprehensive and unbiased mapping at the mesoscale to characterize cell reorganization that would be difficult to detect with other existing methodologies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Mitochondria are cellular organelles responsible for energy production, and dysregulation of the mitochondrial network is associated with many disease states. To fully characterize the mitochondrial network's structure and function, a three-dimensional whole cell mapping technique is required., Scope of Review: This review highlights the use of soft X-ray tomography (SXT) as a relatively high-throughput approach to quantify mitochondrial structure and function under multiple cellular conditions., Major Conclusions: The use of SXT opens the door for mapping cellular rearrangements during critical processes such as insulin secretion, stem cell differentiation, or disease progression. SXT provides unique information such as biochemical compositions or molecular densities of organelles and allows for unbiased, label-free imaging of intact whole cells. Mapping mitochondria in the context of the near-native cellular environment will reveal more information regarding mitochondrial network functions within the cell., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The protocol describes step-by-step sample preparation, data acquisition, and segmentation of cellular organelles with soft X-ray tomography. It is designed for microscopes built to perform full-rotation data acquisition on specimens in cylindrical sample holders, such as the XM-2 microscope at the Advanced Light Source, LBNL; however, it might be generalized for similar sample holder designs for both synchrotron and table-top microscopes. For complete details on the use and execution of this profile, please refer to Loconte et al. (2021)., Competing Interests: The authors declare no competing interests, (© 2022 The Author(s).)

Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

High-resolution and rapid imaging of host cell ultrastructure can generate insights toward viral disease mechanism, for example for a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Here, we employ full-rotation soft X-ray tomography (SXT) to examine organelle remodeling induced by SARS-CoV-2 at the whole-cell level with high spatial resolution and throughput. Most of the current SXT systems suffer from a restricted field of view due to use of flat sample supports and artifacts due to missing data. In this approach using cylindrical sample holders, a full-rotation tomogram of human lung epithelial cells is performed in less than 10 min. We demonstrate the potential of SXT imaging by visualizing aggregates of SARS-CoV-2 virions and virus-induced intracellular alterations. This rapid whole-cell imaging approach allows us to visualize the spatiotemporal changes of cellular organelles upon viral infection in a quantitative manner., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.

In the lungs, asbestos develops an Fe-rich coating (Asbestos Body, AB) that becomes the actual interface between the foreign fibers and the host organism. Conventional approaches to study ABs require an invasive sample preparation that can alter them. In this work, a novel combination of x-ray tomography and spectroscopy allowed studying unaltered lung tissue samples with chrysotile and crocidolite asbestos. The thickness and mass density maps of the ABs obtained by x-ray tomography were used to derive a truly quantitative elemental analysis from scanning x-ray fluorescence spectroscopy data. The average mass density of the ABs is compatible with that of highly loaded ferritin, or hemosiderin. The composition of all ABs analyzed was similar, with only minor differences in the relative elemental fractions. Silicon concentration decreased in the core-to-rim direction, indicating a possible partial dissolution of the inner fiber. The Fe content in the ABs was higher than that possibly contained in chrysotile and crocidolite. This finding opens two opposite scenarios, the first with Fe coming from the fiber bulk and concentrating on the surface as long as the fiber dissolves, the second where the Fe that takes part to the formation of the AB originates from the host organism Fe-pool., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Correlation of 3D images acquired on different microscopes can be a daunting prospect even for experienced users. This protocol describes steps for registration of images from soft X-ray absorption contrast imaging and super-resolution fluorescence imaging of hydrated biological materials at cryogenic temperatures. Although it is developed for data generated at synchrotron beamlines that offer the above combination of microscopies, it is applicable to all analogous imaging systems where the same area of a sample is examined using successive non-destructive imaging techniques. For complete details on the use and execution of this protocol, please refer to Kounatidis et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

A high prevalence of obstructive sleep apnea (OSA) has been reported in Down syndrome (DS) owing to the coexistence of multiple predisposing factors related to its genetic abnormality, posing a challenge for the management of OSA. We hypothesized that DS mice recapitulate craniofacial abnormalities and upper airway obstruction of human DS and can serve as an experimental platform for OSA research. This study, thus, aimed to quantitatively characterize the upper airway as well as craniofacial abnormalities in Dp(16)1Yey (Dp16) mice. Dp16 mice demonstrated craniofacial hypoplasia, especially in the ventral part of the skull and the mandible, and rostrally positioned hyoid. These changes were accompanied with a shorter length and smaller cross-sectional area of the upper airway, resulting in a significantly reduced upper airway volume in Dp16 mice. Our non-invasive approach, a combination of computational fluid dynamics and high-resolution micro-CT imaging, revealed a higher negative pressure inside the airway of Dp16 mice compared to wild-type littermates, showing the potential risk of upper airway collapse. Our study indicated that Dp16 mice can be a useful model to examine the pathophysiology of increased upper airway collapsibility of DS and to evaluate the efficacy of therapeutic interventions for breathing and sleep anomalies.

Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes has been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, while insightful, are nonetheless limited either by restrictions in resolution or by the sheer complexity of generating multidimensional data. Recent advances in the use and application of X-rays to acquire micro- and nanotomographic data sets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the subcellular analyses of unstained vitrified cells and three-dimensional localization of specific protein targets and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles, and applications of both soft X-ray tomography and X-ray microscopy and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches and detail how they have become integral in correlative microscopy workflows., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Bayguinov et al.)

Laparoscopic Sleeve gastrectomy (LSG) is the most commonly performed bariatric surgical procedure worldwide. There is wide variation however in post-operative weight loss on long term follow-up, and residual gastric volume (RGV) is believed to be an important variable. Multiple studies have correlated RGV as assessed by Computerized Tomography volumetry with excess weight loss (EWL%) following LSG, but definite consensus is lacking. This article systematically reviews the published studies in English literature to ascertain whether any correlation exists between the RGV and EWL% following LSG. Ten studies were included in this review, and significant differences were noted in the technique of RGV assessment, and timing of RGV and EWL% assessment. Five studies found a statistically significant correlation between the RGV and EWL%. One study found a correlation which did not reach statistical significance. Two additional studies reported that the resected volume rather than RGV correlated with the EWL%. Meta-analysis of studies reporting correlation between RGV and EWL% showed that up to 26.3% (95% CI: 5.1%-56.1%) of variability in EWL% can be explained by variations in RGV. A lower RGV is likely to result in a better post-operative weight loss following LSG. There is need for standardization of technique and timing of RGV assessment., (© 2020 World Obesity Federation.)

Context: A common genetic variant near PPP1R3B (rs4841132G > A) has been associated with increased hepatic computed tomography (CT) attenuation and with plasma levels of glucose and liver enzymes., Objective: To elucidate the association of rs4841132 with hepatic CT attenuation, and to test if synergistic effects modify the association of the variant with plasma glucose and liver enzymes., Design: Population-based cohort study., Setting and Participants: The Copenhagen City Heart Study and the Copenhagen General Population Study combined, totaling 107 192 individuals from the Danish general population. Hepatic CT scans were available in 6445 individuals., Main Outcome Measures: Hepatic CT attenuation and plasma levels of glucose and liver enzymes., Results: The rs4841132 A-allele (rs4841132-A) was associated with higher hepatic CT attenuation (P = 5 × 10-6). The probability of carrying rs4841132-A increased with higher hepatic CT attenuation in the range above 65 Hounsfield units, but remained constant at the range below (P = 4 × 10-8 for nonlinearity). Rs4841132-A was associated with up to 0.17 mmol/L higher plasma glucose in fasting individuals, but with up to 0.17 mmol/L lower glucose in postprandial individuals (P = 6 × 10-5 for interaction between rs4841132 and time since last meal on plasma glucose). Finally, rs4841132-A was associated with up to 2 U/L higher plasma alanine transaminase (P = 3 × 10-6). This association was not modified by adiposity, alcohol intake, or steatogenic genetic risk., Conclusions: Rs4841132-A associates with higher hepatic CT attenuation in a distinctly nonlinear manner, and its association with plasma glucose depends on prandial status. The overall association pattern supports that rs4841132-A promotes hepatic glycogen synthesis postprandially., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alzheimer's disease (AD) is a neurodegenerative disorder which is characterized by increasing dementia. It is accompanied by the development of extracellular β-amyloid plaques and neurofibrillary tangles in the gray matter of the brain. Histology is the gold standard for the visualization of this pathology, but also has intrinsic shortcomings. Fully three-dimensional analysis and quantitative metrics of alterations in the tissue structure require a complementary approach. In this work we use x-ray phase-contrast tomography to obtain three-dimensional reconstructions of human hippocampal tissue affected by AD. Due to intrinsic electron density differences, tissue components and structures such as the granule cells of the dentate gyrus, blood vessels, or mineralized plaques can be identified and segmented in large volumes. Based on correlative histology, protein (tau, β-amyloid) and elemental content (iron, calcium) can be attributed to certain morphological features occurring in the entire volume. In the vicinity of senile plaques, an accumulation of microglia in combination with a loss of neuronal cells can be observed., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Imaging techniques for quantifying changes in the hierarchical structure of deforming joints are constrained by destructive sample treatments, sample-size restrictions and lengthy scan times. Here, we report the use of fast low-dose pink-beam synchrotron X-ray tomography in combination with mechanical loading at nanometric precision for in situ imaging, at resolutions below 100 nm, of the mechanical strain in intact untreated joints under physiologically realistic conditions. We show that in young, older and osteoarthritic mice, hierarchical changes in tissue structure and mechanical behaviour can be simultaneously visualized, and that the tissue structure at the cellular level correlates with the mechanical performance of the whole joint. We also use the tomographic approach to study the colocalization of tissue strains to specific chondrocyte lacunar organizations within intact loaded joints and to explore the role of calcified-cartilage stiffness on the biomechanics of healthy and pathological joints.

X-ray tomography has become an indispensable tool for studying complex 3D interior structures with high spatial resolution. Three-dimensional imaging using soft X-rays offers powerful contrast mechanisms but has seen limited success with tomography due to the restrictions imposed by the much lower energy of the probe beam. The generalized geometry of laminography, characterized by a tilted axis of rotation, provides nm-scale 3D resolution for the investigation of extended (mm range) but thin (μm to nm) samples that are well suited to soft X-ray studies. This work reports on the implementation of soft X-ray laminography (SoXL) at the scanning transmission X-ray spectromicroscope of the PolLux beamline at the Swiss Light Source, Paul Scherrer Institut, which enables 3D imaging of extended specimens from 270 to 1500 eV. Soft X-ray imaging provides contrast mechanisms for both chemical sensitivity to molecular bonds and oxidation states and magnetic dichroism due to the much stronger attenuation of X-rays in this energy range. The presented examples of applications range from functionalized nanomaterials to biological photonic crystals and sophisticated nanoscaled magnetic domain patterns, thus illustrating the wide fields of research that can benefit from SoXL.

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics., Competing Interests: The authors have declared that no competing interests exist.

The iridium half-sandwich complex [Ir(η 5 :κ 1 -C 5 Me 4 CH 2 py)(2-phenylpyridine)]PF 6 is highly cytotoxic: 15-250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X-ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X-ray tomography (cryo-SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X-ray fluorescence tomography (cryo-XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo-3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Globally, lung cancer is one of the most frequently diagnosed and deadliest types of cancer. Lung cancer imaging can be performed using both invasive and noninvasive techniques, including magnetic resonance, positron emission tomography, single photon emission computed tomography, chest radiography, and computed tomography. But nonspecific contrast agents and radiopharmaceuticals are insufficient for early and specific diagnoses and imaging. In the case of lung cancer therapy, conventional therapeutic agents and radiotherapy may cause severe and systemic adverse and toxic effects and fail to eradicate all tumor tissue. Therefore, formulation of novel, targeted, and specific agents is critically important to overcome these challenges. In this review, we summarize lung cancer classification, current methods for lung cancer imaging and therapy, and future options containing nanosized systems for lung cancer imaging and/or therapy.

Objective: Pleomorphic adenoma is the most common benign salivary gland neoplasm in children. Recurrence and malignant transformation are concerns necessitating proper treatment. The goal of this research was to discuss the presentation, evaluation, and management of pediatric pleomorphic adenoma of the head and neck., Methods: Retrospective chart review of patients under 20 years of age treated for pleomorphic adenoma of the head and neck between 1998 and 2017. Data assessed included demographics, clinical presentation, imaging, treatment, complications, recurrence, and follow-up., Results: Forty-one patients with pleomorphic adenoma were identified. Major salivary gland lesions were most common (n = 32, 78.0%); 78.1% (25 of 32) arising in the parotid and 21.9% (7 of 32) in the submandibular glands. Minor salivary gland lesions were removed from the palate (5 of 9, 55.6%), buccal mucosa (2 of 9, 22.2%), parapharyngeal space (1 of 9, 11.1%), and upper lip (1 of 9, 11.1%). Preoperative imaging was reviewed in 32 patients and consisted of nine ultrasound exams, 15 computerized tomography (CT) exams, and 23 magnetic resonance imaging (MRI) exams. Multimodality imaging was performed in 14 of 32 patients. Surgical excision was performed in all patients. Postoperative complications included transient facial paresis (n = 9), Frey syndrome (n = 1), and bacterial pharyngitis (n = 1). Average length of follow-up was 25.5 months; confirmed recurrence occurred in two patients (4.9%)., Conclusion: Evaluation of pleomorphic adenoma in children should include preoperative imaging. The goal of surgery should be complete surgical excision with negative margins. Rate of recurrence is low; however, prolonged monitoring is recommended., Level of Evidence: 4. Laryngoscope, 129:2603-2609, 2019., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Cellular complexity is represented best in high-spatial resolution, three-dimensional (3D) reconstructions. Soft X-ray tomography (SXT) generates detailed volumetric reconstructions of cells preserved in a near-to-native, frozen-hydrated state. SXT is broadly applicable and can image specimens ranging from bacteria to large mammalian cells. As a reference, we summarize light and electron microscopic methods. We then present an overview of SXT and discuss its role in cellular imaging. We detail the methods used to image biological specimens and present recent highlights that illustrate the capabilities of the technique. We conclude by discussing correlative imaging, specifically the combination of SXT and fluorescence microscopy performed on the same specimen. This correlated approach combines the structural morphology of a cell with its physiological characteristics to build a deeply informative composite view., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

X-ray computed tomography is a strong tool that finds many applications both in medical applications and in the investigation of biological and nonbiological samples. In the clinics, X-ray tomography is widely used for diagnostic purposes whose three-dimensional imaging in high resolution helps physicians to obtain detailed image of investigated regions. Researchers in biological sciences and engineering use X-ray tomography because it is a nondestructive method to assess the structure of their samples. In both medical and biological applications, visualization of soft tissues and structures requires special treatment, in which special contrast agents are used. In this detailed report, molecule-based and nanoparticle-based contrast agents used in biological applications to enhance the image quality were compiled and reported. Special contrast agent applications and protocols to enhance the contrast for the biological applications and works to develop nanoparticle contrast agents to enhance the contrast for targeted drug delivery and general imaging applications were also assessed and listed., (© 2019 Wiley Periodicals, Inc.)

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can provide unrivalled high-resolution images of specific features and volumes of interest. However, the regions interrogated are typically very small, and sample preparation is both time-consuming and destructive. Here we consider how prior X-ray micro-computed tomography (microCT) presents an opportunity to increase the efficiency of electron microscopy in biology. We demonstrate how it can be used to; select the most promising samples and target site-specific locations; provide a wider context of the location being interrogated (multiscale correlative imaging); guide sample preparation and 3D imaging schemes; as well as quantify the effects of destructive sample preparation and staining procedures. We present a workflow utilising open source software in which microCT can be used either broadly, or precisely, to experimentally steer and inform subsequent electron microscopy studies. As automated sample registration procedures are developed to enable correlative microscopy, experimental steering by prior CT could be beneficially routinely incorporated into many experimental workflows., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Morphometric measurements, such as quantifying cell shape, characterizing sub-cellular organization, and probing cell-cell interactions, are fundamental in cell biology and clinical medicine. Until quite recently, the main source of morphometric data on cells has been light- and electron-based microscope images. However, many technological advances have propelled X-ray microscopy into becoming another source of high-quality morphometric information. Here, we review the status of X-ray microscopy as a quantitative biological imaging modality. We also describe the combination of X-ray microscopy data with information from other modalities to generate polychromatic views of biological systems. For example, the amalgamation of molecular localization data, from fluorescence microscopy or spectromicroscopy, with structural information from X-ray tomography. This combination of data from the same specimen generates a more complete picture of the system than that can be obtained by a single microscopy method. Such multimodal combinations greatly enhance our understanding of biology by combining physiological and morphological data to create models that more accurately reflect the complexities of life., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The ESCRT machinery mediates scission of the intercellular bridge that connects two daughter cells at the end of cytokinesis. Structured illumination microscopy (SIM) and cryo-soft-X-ray tomography (cryo-SXT) have been used in recent years to study the topology of ESCRT-driven cytokinetic abscission. These studies revealed that the intercellular bridge is occupied by cortical rings and spiral-like filaments and that ESCRTs form ring-like structures in this region during abscission. In this chapter, we provide two protocols: a protocol for determining the spatial organization of specific ESCRT components at the intercellular bridge using SIM and a protocol for resolving the ultrastructural organization of cortical filaments at the intercellular bridge using cryo-SXT.

In this article, we introduce a linear approximation of the forward model of soft X-ray tomography, such that the reconstruction is solvable by standard iterative schemes. This linear model takes into account the three-dimensional point spread function (PSF) of the optical system, which consequently enhances the reconstruction of data. The feasibility of the model is demonstrated on both simulated and experimental data, based on theoretically estimated and experimentally measured PSFs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

X-ray Fluorescence (XRF) microscopy is a growing approach for imaging the trace element concentration, distribution, and speciation in biological cells at the nanoscale. Moreover, three-dimensional nanotomography provides the added advantage of imaging subcellular structure and chemical identity in three dimensions without the need for staining or sectioning of cells. To date, technical challenges in X-ray optics, sample preparation, and detection sensitivity have limited the use of XRF nanotomography in this area. Here, XRF nanotomography was used to image the elemental distribution in individual E. coli bacterial cells using a sub-15 nm beam at the Hard X-ray Nanoprobe beamline (HXN, 3-ID) at NSLS-II. These measurements were simultaneously combined with ptychography to image structural components of the cells. The cells were embedded in small (3-20 µm) sodium chloride crystals, which provided a non-aqueous matrix to retain the three-dimensional structure of the E. coli while collecting data at room temperature. Results showed a generally uniform distribution of calcium in the cells, but an inhomogeneous zinc distribution, most notably with concentrated regions of zinc at the polar ends of the cells. This work demonstrates that simultaneous two-dimensional ptychography and XRF nanotomography can be performed with a sub-15 nm beam size on unfrozen biological cells to co-localize elemental distribution and nanostructure simultaneously.

We have investigated limited angle transmission tomography to estimate speed of sound (SOS) distributions for breast cancer detection. That requires both accurate delineations of major tissues, in this case by segmentation of prior B-mode images, and calibration of the relative positions of the opposed transducers. Experimental sensitivity evaluation of the reconstructions with respect to segmentation and calibration errors is difficult with our current system. Therefore, parametric studies of SOS errors in our bent-ray reconstructions were simulated. They included mis-segmentation of an object of interest or a nearby object, and miscalibration of relative transducer positions in 3D. Close correspondence of reconstruction accuracy was verified in the simplest case, a cylindrical object in homogeneous background with induced segmentation and calibration inaccuracies. Simulated mis-segmentation in object size and lateral location produced maximum SOS errors of 6.3% within 10 mm diameter change and 9.1% within 5 mm shift, respectively. Modest errors in assumed transducer separation produced the maximum SOS error from miscalibrations (57.3% within 5 mm shift), still, correction of this type of error can easily be achieved in the clinic. This study should aid in designing adequate transducer mounts and calibration procedures, and in specification of B-mode image quality and segmentation algorithms for limited angle transmission tomography relying on ray tracing algorithms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

High-spatial-resolution histology of coronary artery autopsy samples play an important role for understanding heart disease such as myocardial infarction. Unfortunately, classical histology is often destructive, has thick slicing, requires extensive sample preparation, and is time-consuming. X-ray micro-CT provides fast nondestructive 3D imaging but absorption contrast is often insufficient, especially for observing soft-tissue features with high resolution. Here we show that propagation-based x-ray phase-contrast tomography has the resolution and contrast to image clinically relevant soft-tissue features in intact coronary artery autopsy samples with cellular resolution. We observe microscopic lipid-rich plaques, individual adipose cells, ensembles of few foam cells, and the thin fibrous cap. The method relies on a small-spot laboratory x-ray microfocus source, and provides high-spatial resolution in all three dimensions, fast data acquisition, minimum sample distortion and requires no sample preparation.

Soft x-ray microscopy (SXM) is a minimally invasive technique for single-cell high-resolution imaging as well as the visualization of intracellular distributions of light elements such as carbon, nitrogen, and oxygen. We used SXM to observe photosynthesis and nitrogen fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120, which can form heterocysts during nitrogen starvation. Statistical and spectroscopic analyses from SXM images around the K-absorption edge of nitrogen revealed a significant difference in the carbon-to-nitrogen (C/N) ratio between vegetative cells and heterocysts. Application of this analysis to soft x-ray images of Anabaena sp. PCC 7120 revealed inhomogenous C/N ratios in the cells. Furthermore, soft x-ray tomography of Anabaena sp. PCC 7120 revealed differing cellular C/N ratios, indicating different carbon and nitrogen distributions between vegetative cells and heterocysts in three dimensions., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Dual-energy X-ray absorptiometry (DXA) as currently used has limitations in identifying patients with osteoporosis and predicting occurrence of fracture. We aimed to express peripheral quantitative computed tomography (pQCT) variables of patients with low-trauma fracture as T-scores by using T-score scales obtained from healthy young women, and to evaluate the potential clinical utility of pQCT for the assessment of bone fragility. Fracture patients were recruited from a fracture liaison service at the Royal Melbourne Hospital. Reference pQCT data were obtained from studies on women's health conducted by our group. A study visit was arranged with fracture patients, during which DXA and pQCT were applied to measure their bone strength. A total of 59 fracture patients were recruited, and reference data were obtained from 78 healthy young females. All DXA variables and most pQCT variables were significantly different between healthy young females and fracture patients (p < 0.05), except polar stress-strain index (p = 0.34) and cortical bone density (p = 0.19). Fracture patients were divided into osteoporosis and non-osteoporosis groups according to their DXA T-scores. Significant differences were observed in most pQCT variables (p < 0.05), except trabecular area and cortical density (p > 0.9 and p = 0.5, respectively). By applying pQCT T-scores, 11 (27%) of patients who were classified as having low or medium risk of osteoporosis on DXA T-scores alone were reclassified as high risk. Results of logistic regression suggested trabecular bone density as an independent predictor of osteoporosis status. More patients can be identified with osteoporosis by applying pQCT T-score variables in older people with low-trauma fracture. Peripheral QCT T-scores contribute to the understanding of bone fragility in this population., (Copyright © 2017 The International Society for Clinical Densitometry. All rights reserved.)

In order to advance the state-of-the-art in computational aortic biomechanics, we investigated the influence of (i) a non-uniform wall thickness, (ii) minor aortic side branches and (iii) a non-uniform axial stretch distribution on the location of predicted hotspots of principal strain in a mouse model for dissecting aneurysms. After 3 days of angiotensin II infusion, a murine abdominal aorta was scanned in vivo with contrast-enhanced micro-CT. The animal was subsequently sacrificed and its aorta was scanned ex vivo with phase-contrast X-ray tomographic microscopy (PCXTM). An automatic morphing framework was developed to map the non-pressurized, non-stretched PCXTM geometry onto the pressurized, stretched micro-CT geometry. The output of the morphing model was a structural FEM simulation where the output strain distribution represents an estimation of the wall deformation, not only due to the pressurization, but also due to the local axial stretch field. The morphing model also included minor branches and a mouse-specific wall thickness. A sensitivity study was then performed to assess the influence of each of these novel features on the outcome of the simulations. The results were supported by comparing the computed hotspots of principal strain to hotspots of early vascular damage as detected on PCXTM. Non-uniform axial stretch, non-uniform wall thickness and minor subcostal arteries significantly alter the locations of calculated hotspots of maximal principal strain. Even if experimental data on these features are often not available in clinical practice, one should be aware of the important implications that simplifications in the model might have on the final simulated result.