Correlative Cryo-imaging Using Soft X-Ray Tomography for the Study of Virus Biology in Cells and Tissues.

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.

Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

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.

Dehydration as alternative sample preparation for soft X-ray tomography.

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.

3D surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semisupervised deep learning.

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.

Radiation Dose Reduction in Contrast-Enhanced Abdominal CT: Comparison of Photon-Counting Detector CT with 2nd Generation Dual-Source Dual-Energy CT in an oncologic cohort.

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 2nd-generation DSCT and at follow-up on a 1st-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 (CTDIvol), 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). CNRvessel and SNRvessel 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 2nd-generation DSCT.

Soft X-ray tomograms provide a structural basis for whole-cell modeling.

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.

Neumoencéfalo recurrente post quirúrgico, a raíz de dos casos / Post-surgical late pneumoencephalus, based on two cases

El neumoencéfalo es una patología que comúnmente se presenta después de cirugía neuroquirúrgica y ocasionalmente endonasal. Estos se suelen manejar de manera conservadora, sin embargo, se pueden asociar a distintas etiologías las cuales los hacen recurrir. En este reporte presentamos dos casos de neumoencéfalo tardío post quirúrgico asociado a fístulas de LCR de bajo flujo, donde se discute su clínica, etiología y manejo posterior.

Pneumocephalus is a pathology that commonly occurs after endonasal surgery, these are usually managed conservatively, however they can be associated with different etiologies which make them recur. In this report we present two cases of post-surgical late pneumocephalus associated with low-flow CSF fistulae, where its symptoms, etiology, and subsequent management are discussed.

Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells.

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.

Evaluation of the Role of Bacterial Amyloid on Nucleoid Structure Using Cryo-Soft X-Ray Tomography.

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.

Shock séptico secundario a fascitis necrotizante abdominal tras drenaje de absceso interesfintérico perianal / Septic shock secondary to abdominal necrotizing fascitis after drainage of perianal intersphyteric abscess

Resumen Objetivo: El objetivo de este manuscrito es presentar el caso de un varón de 41 años que debuta con shock séptico y fascitis necrotizante abdominal en el posoperatorio del desbridamiento de un absceso perianal para focalizar la atención del lector en la posible evolución clínica hacia gangrena de Fournier. Materiales y Método: Tras la intervención, el paciente refiere aumento de temperatura y sensación de crepitación subcutánea a nivel abdominal, junto con empeoramiento clínico y hemodinámico, evidenciándose evolución tórpida hacia gangrena de Fournier extendida a región abdominal. Resultados: Tras la reintervención, el paciente presentó una evolución favorable aunque requirió sucesivas curas y desbridamientos quirúrgicos. Conclusiones y Discusión: Cabe destacar la importancia de una exploración clínica completa y detallada previa a cualquier intervención quirúrgica, así como el diagnóstico temprano en situaciones de shock séptico que permitan inicio de antibioterapia precoz y control del foco eficaz.

Aim: The objective of this manuscript is to present the case of a 41-year-old man with septic shock and abdominal necrotizing fasciitis after drainage of an interesphinteric perianal abscess to focus the reader's attention on the possible clinical evolution towards Fournier's gangrene. Materials and Method: After the intervention, the patient reported an increase in temperature and a sensation of subcutaneous crepitus at the abdominal level, with clinical and hemodynamic worsening, showing a torpid evolution towards Fournier's gangrene extended to the abdominal area. Results: After the reoperation, the patient presented a favorable evolution, although he required successive cures and surgical debridements. Conclusions and Discussion: It is worth highlighting the importance of a complete and detailed clinical examination prior to any surgical intervention, as well as the early diagnosis in situations of septic shock that allow early initiation of antibiotic therapy and effective control of the focus.

Sacroileítis infecciosa secundaria a un absceso del iliopsoas ­ reporte de un caso / Infectious sacroiliitis secondary to an iliopsoas abscess ­ a case report

La sacroileítis infecciosa (SII), también descrita en la literatura como sacroileítis séptica o piógena, es una patología infrecuente, y su diagnóstico constituye un reto debido a su rareza relativa y la diversa presentación clínica, que frecuentemente imita otros trastornos más prevalentes originados en estructuras vecinas. Se requiere un alto índice de sospecha y un examen físico acucioso para un diagnóstico oportuno, mientras que los estudios de laboratorio y de imagen ayudan a confirmar el diagnóstico y dirigir la estrategia de tratamiento apropiada para evitar complicaciones y secuelas a corto y mediano plazos. Presentamos un caso de paciente de género femenino de 36 años, con cuadro clínico de SII izquierda, secundaria a un absceso del músculo iliopsoas, condición que generalmente se presenta como una complicación de la infección. Se realizaron los diagnósticos clínico, imagenológico y Biológico, se inició el tratamiento antibiótico oportuno, y se logró una excelente evolución clínica, sin secuelas

Infectious sacroiliitis (ISI), also described in the literature as septic or pyogenic sacroiliitis, is an infrequent pathology, and its diagnosis constitutes a challenge due to its relative rarity and the diverse clinical presentation, frequently imitating other more prevalent disorders originating in neighboring structures. A high index of suspicion and a thorough physical examination are required in order to establish an opportune diagnosis, while laboratory and imaging studies help confirm the diagnosis and direct the appropriate treatment strategy to avoid complications and sequelae in the short and medium terms. We herein present a case of a female patient aged 36 years, with a clinical picture of left ISI, secondary to an iliopsoas muscle abscess, a condition that usually presents as a complication of the infection. The clinical, imaging and microbiological diagnoses were made, the timely antibiotic treatment was initiated, and an excellent clinical evolution without sequelae was achieved.

A guide into the world of high-resolution 3D imaging: the case of soft X-ray tomography for the life sciences.

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.

Soft X-ray tomography to map and quantify organelle interactions at the mesoscale.

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 ß 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.

A protocol for full-rotation soft X-ray tomography of single cells.

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).

The use of soft X-ray tomography to explore mitochondrial structure and function.

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.

Using soft X-ray tomography for rapid whole-cell quantitative imaging of SARS-CoV-2-infected cells.

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.

Imaging of Virus-Infected Cells with Soft X-ray Tomography.

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.

Fístula biliobronquial secundaria a quiste hidatídico hepático en tránsito torácico: caso clínico / Bronchobiliary fistula secondary to hepatic hydatid cyst in thoracic transit: clinical case

Resumen Introducción: En un quiste hidatídico hepático pueden ocurrir una serie de complicaciones de diversa gravedad. Una es el tránsito hepatotorácico (THT), que es el compromiso simultáneo de hígado, diafragma y pulmón secundario a migración de un quiste hidatídico hepático. Objetivo: Presentar una complicación de baja incidencia de un quiste hidatídico hepático como lo es el THT con fistula biliobronquial y posterior resolución quirúrgica. Materiales y Método: Registro clínico, imagenológico y fotográfico del episodio clínico. Resultados: Paciente con diagnóstico de quiste hidatídico en tránsito hepatotorácico con fístula biliobronquial fue sometida a toracotomía, resección en cuña del pulmón incluyendo bronquios comunicantes con el quiste. Paciente presenta evolución clínica e imagenológica favorable. Discusión: Se discuten formas de presentación, complicaciones de la evolución, grados de progresión y ubicaciones anatómicas frecuentes. Se hace énfasis en rol de la clínica e imagenología para diagnóstico y lo controversial del manejo. Conclusión: Tratamiento quirúrgico dependerá de localización de la lesión, estado del quiste, tamaño y experiencia del equipo quirúrgico, siendo una quistectomía con tratamiento de los trayectos fistulosos una buena alternativa.

Introduction: A variety of severe complications can occur in a hepatic hydatid cyst. One of them is the transit from liver to thorax through the diaphragm (HTT). Aim: To present a low impact complication of a hepatic hydatid cyst such as HTT with bronchobiliary fistula and subsequent surgical procedure. Materials and Method: Clinical, imaging, and photographic record of the clinical event. Results: A patient with a diagnosis of hydatid cyst in hepatothoracic transit with bronchobiliary fistula underwent thoracotomy with a wedge resection of the lung, including a cyst-bronchial communication. The patient presents good clinical and imaging evolution. Discussion: Forms of presentation, evolutionary complications, stages of progression, and frequent anatomical locations are discussed. Emphasis is made on the role of the clinical examination and imaging tests for diagnosis and controversial management. Conclusión: Surgical treatment will depend on location of the lesion, experience of the surgical team, condition and size of the cyst, being a cystectomy with the treatment of the fistulous tracts a good alternative.

Protocol for image registration of correlative soft X-ray tomography and super-resolution structured illumination microscopy images.

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).

Chemo-physical properties of asbestos bodies in human lung tissues studied at the nano-scale by non-invasive, label free x-ray imaging and spectroscopic techniques.

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.