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.

High-speed rotating device for X-ray tomography with 10 ms temporal resolution.

The temporal resolution of X-ray tomography, using a synchrotron radiation X-ray source, has been improved to millisecond order in recent years. However, the sample must be rotated at a speed of more than a few thousand revolutions per minute, which makes it difficult to control the environment around the sample. In this study, a high-speed rotation device has been developed, comprising two synchronized coaxial motors movable along the direction of the axis, which can stretch or compress the rotating sample. Using this device, tomograms of breaking rubber were successfully obtained at a temporal resolution of 10 ms.

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches.

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.

Switchable resolution in soft x-ray tomography of single cells.

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.

Correlative x-ray phase-contrast tomography and histology of human brain tissue affected by Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder which is characterized by increasing dementia. It is accompanied by the development of extracellular ß-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, ß-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.

X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam.

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.

OMNY-A tOMography Nano crYo stage.

For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved via dedicated laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy. Cryogenic sample conditions are maintained via conductive cooling. 90 K can be reached when using liquid nitrogen as coolant, and 10 K is possible with liquid helium. A cryogenic sample-change mechanism permits measurements of cryogenically fixed specimens. We compare images obtained with OMNY with older measurements performed using a nitrogen gas cryo-jet of stained, epoxy-embedded retina tissue and of frozen-hydrated Chlamydomonas cells.

Lensless Tomographic Imaging of Near Surface Structures of Frozen Hydrated Malaria-Infected Human Erythrocytes by Coherent X-Ray Diffraction Microscopy.

Lensless, coherent X-ray diffraction microscopy has been drawing considerable attentions for tomographic imaging of whole human cells. In this study, we performed cryogenic coherent X-ray diffraction imaging of human erythrocytes with and without malaria infection. To shed light on structural features near the surface, "ghost cells" were prepared by the removal of cytoplasm. From two-dimensional images, we found that the surface of erythrocytes after 32 h of infection became much rougher compared to that of healthy, uninfected erythrocytes. The Gaussian roughness of an infected erythrocyte surface (69 nm) is about two times larger than that of an uninfected one (31 nm), reflecting the formation of protein knobs on infected erythrocyte surfaces. Three-dimensional tomography further enables to obtain images of the whole cells with no remarkable radiation damage, whose accuracy was estimated using phase retrieval transfer functions to be as good as 64 nm for uninfected and 80 nm for infected erythrocytes, respectively. Future improvements in phase retrieval algorithm, increase in degree of coherence, and higher flux in combination with complementary X-ray fluorescence are necessary to gain both structural and chemical details of mesoscopic architectures, such as cytoskeletons, membraneous structures, and protein complexes, in frozen hydrated human cells, especially under diseased states.

CT scan prior to radiotherapy in unresectable, locally advanced, non-small cell carcinoma of the lung: is it always necessary?

Purpose. There is broad consensus regarding evaluating response to chemotherapy (CHT) by means of computerized tomography (CT) in patients with localized or locally advanced non-small cell lung carcinoma (NSCLC). We present a study comparing the usefulness of CT versus chest X-ray (XR) and clinical findings when indicating radiotherapy (RT) following CHT. Methods. Ninety-eight of 150 subjects with unresectable locally advanced NSCLC were blindly and independently evaluated by XR and CT, with pairs of chest XR and CT (before and after CHT). A null hypothesis (H0) was established of the conditioned probability of detecting progression by CT and not by XR of 10 % or more, with a statistical power of 80 %. Results. Sensitivity, specificity, positive and negative predictive value of XR versus CT were 98, 89, 99, and 80 % respectively. A 4 % (p = 0.0451) probability of improvement of CT versus XR was calculated, enabling the H0 to be ruled out. Conclusion. The CT failed to prove to be significantly superior to the chest XR + clinical picture in indicating a change in treatment approach in patients with unresectable locally advanced NSCLC after CHT (AU)

No disponible

Abordagem multidisciplinar no diagnóstico da respiração bucal / Multidisciplinary approach in the diagnosis of mouth breathing

A respiração bucal se caracteriza por uma obstrução das vias aéreas superiores que leva o paciente ao hábitode respirar pela boca. Como consequência, ocorre uma adaptação postural seguida por modificações nasarcadas dentárias e no crescimento facial, além de infecções e má oxigenação cerebral. O objetivo desse artigo foi apresentar os aspectos relacionados ao diagnóstico da respiração bucal e as competências da equipe multidisciplinar, com enfoque na odontologia/ortodontia. A revisão de literatura foi feita em resenhas e artigospublicados entre 1936 a 2015, livros e nos sítios eletrônicos Lilacs, Scielo, Cochrane, Medline, Pubmed e Bireme. Pôde-se concluir que é uma síndrome multifatorial e, para o êxito no seu diagnóstico, há necessidadede interação e atuação de profissionais especializados em diversas áreas, por isso o grande interesse das áreasmédica, odontológica e fonoaudiológica

Mouth breathing is characterized by an obstruction of the upper airway that leads the patient to the habit ofbreathing through the mouth. As a result, there is a postural adjustment, which is followed by changes indental arches and then the facial growth, infections and poor brain oxygenation. The purpose of this paper wasto present aspects related to the diagnosis of mouth breathing and the skills of a multidisciplinary team, with afocus on dentistry / orthodontics. The literature review was made in reviews and articles published between1936-2015, books and electronic sites Lilacs, SciELO, Cochrane, Medline, Pubmed and Bireme. It was concludedthat it is a multifactorial syndrome and for the successful diagnosis, it is necessary the interaction andproceeding of professionals specialized in different areas, therfore the great interest of the medical, dental andspeech areas

Impact of New-generation Hybrid Imaging Technology on Radiation Dose during Percutaneous Coronary Interventions and Trans-femoral Aortic Valve Implantations: A comparison with conventional flat-plate angiography.

BACKGROUND: Technological advancements in newer-generation catheterisation laboratories may reduce patient and occupational radiation exposure. METHODS: We compared fluoroscopy time and dose-area product (DAP) between a Philips Allura X-PER FD20 and Siemens Artis Zeego Hybrid systems for 47 single-vessel percutaneous coronary interventions (PCI) and 35 transcatheter aortic valve implantations (21 Corevalve, 14 Edwards Sapien TAVI) using the FD20, versus 30 PCI and 28 TAVI (15 Corevalve, 13 Sapien) with the Zeego over a 24-month period. RESULTS: Multivariate analysis revealed that, adjusting for patient weight and fluoroscopy time, DAP (median, interquartile range) was 26% lower for PCI with the Zeego than the FD20 [55.6 (27.0-91.5) vs 77.6 (51.2-129.1) Gy.cm(2), P=0.03)] and using tomographic imaging with the Zeego did not increase DAP for TAVI procedures [98.1 (65.9-136.6) vs 112.4 (64.9-156.2) Gy.cm(2) (P=NS). Although fluoroscopy times were longer for TAVI procedures than PCI with both systems (23.5-24.4 vs 7.3-9.2mins, p<0.0001), there was a significant difference in DAP between PCI and combined TAVI with the Zeego (55.6 vs 112.4Gy.cm(2), P<0.006) but not with the FD20 (77.6 vs 98.1Gy.cm(2), P=NS). CONCLUSION: Specific dose-reducing features of the new-generation system reduced DAP more for PCI than TAVI, as valve replacement procedures use additional cine-acquisition not necessary for PCI.

A versatilidade do uso da tuberosidade maxilar para pequenas reconstruções teciduais na Implantodontia / The versatility of the use of maxillary tuberosity for small tissue reconstructions in Implant Dentistry

As alterações alveolares após a exodontia de um elemento dental desafiam a Implantodontia na busca por técnicas e materiais que devolvam ao rebordo alveolar os tecidos perdidos com previsibilidade e menor morbidade ao paciente. Este relato de casos clínicos mostra a instalação de implantes imediato ou tardio, associados ou não ao procedimento de carga imediata utilizando o enxerto misto da tuberosidade maxilar como alternativa viável para essas reconstruções, tendo como objetivo principal a redução do número de procedimentos cirúrgicos e a previsibilidade no período pós operatório. O osso da tuberosidade maxilar é composto por uma fina camada de osso cortical externo e medular interno que apresentam alta capacidade de revascularização e integração à área receptora. A vantagem em se manter o periósteo entre o enxerto corticomedular do túber e o tecido conjuntivo é conter a reabsorção óssea periosteal na região cervical e melhorar a qualidade do tecido mole. A limitação do uso da tuberosidade maxilar está relacionada a sua dimensão, que pode permitir a reconstrução de um ou dois dentes por área doadora ou, em alguns casos, pode não haver área doadora disponível. As características únicas da tuberosidade maxilar de fornecer osso corticomedular e tecido conjuntivo subepitelial permitem a sua associação no enxerto misto de túber e mostra grande versatilidade para a reconstrução dos tecidos duros e moles, permitindo técnicas refinadas capazes de solucionar casos complexos em um único procedimento cirúrgico...

Alveolar alterations after extraction constitute a challenge and lead to the search for techniques and materials to reconstruct the lost tissues with previsibility and less morbidity to the patients. These case report shows the placement of immediate implants, delayed implants, with or without the immediate loading procedure and maxillary sinus lift with immediate implant placement using the maxillary tuberosity mixed graft as a viable alternative to these reconstructions aiming to reduce the number of surgical procedures and improve predictability in the post-operative period. The tuberosity jaw bone consists of a thin outer layer of cortical bone and inner medullary bone that have a high capacity of revascularization and integrating into the receiving area. The advantage of keeping the periosteum between tuberosity corticomedular graft and the connective tissue is to contain the periosteal bone resorption in the cervical area and improve the quality of the soft tissue. Limitation to the use of maxillary tuberosity is related to their dimensions that may allow the reconstruction of one or two teeth per donor area or, in some cases, it may not be available. The unique characteristics of the maxillary tuberosity to provide corticomedular bone and subepitelial connective tissue allow their association in the maxillary tuberosity mixed graft and show great versatility for the reconstruction of hard and soft tissue, allowing refined techniques to solve complex cases in a single surgical procedure...

Dosimetric characterization and organ dose assessment in digital breast tomosynthesis: Measurements and Monte Carlo simulations using voxel phantoms.

PURPOSE: Due to its capability to more accurately detect deep lesions inside the breast by removing the effect of overlying anatomy, digital breast tomosynthesis (DBT) has the potential to replace the standard mammography technique in clinical screening exams. However, the European Guidelines for DBT dosimetry are still a work in progress and there are little data available on organ doses other than to the breast. It is, therefore, of great importance to assess the dosimetric performance of DBT with respect to the one obtained with standard digital mammography (DM) systems. The aim of this work is twofold: (i) to study the dosimetric properties of a combined DBT/DM system (MAMMOMAT Inspiration Siemens(®)) for a tungsten/rhodium (W/Rh) anode/filter combination and (ii) to evaluate organs doses during a DBT examination. METHODS: For the first task, measurements were performed in manual and automatic exposure control (AEC) modes, using two homogeneous breast phantoms: a PMMA slab phantom and a 4 cm thick breast-shaped rigid phantom, with 50% of glandular tissue in its composition. Monte Carlo (MC) simulations were performed using Monte Carlo N-Particle eXtended v.2.7.0. A MC model was implemented to mimic DM and DBT acquisitions for a wide range of x-ray spectra (24 -34 kV). This was used to calculate mean glandular dose (MGD) and to compute series of backscatter factors (BSFs) that could be inserted into the DBT dosimetric formalism proposed by Dance et al. Regarding the second aim of the study, the implemented MC model of the clinical equipment, together with a female voxel phantom ("Laura"), was used to calculate organ doses considering a typical DBT acquisition. Results were compared with a standard two-view mammography craniocaudal (CC) acquisition. RESULTS: Considering the AEC mode, the acquisition of a single CC view results in a MGD ranging from 0.53 ± 0.07 mGy to 2.41 ± 0.31 mGy in DM mode and from 0.77 ± 0.11 mGy to 2.28 ± 0.32 mGy in DBT mode. Regarding the BSF, the results achieved may lead to a MGD correction of about 6%, contributing to the improvement of the current guidelines used in these applications. Finally, considering the MC results obtained for the organ dose study, the radiation doses found for the tissues of the body other than the breast were in the range of tens of µSv, and are in part comparable to the ones obtained in standard DM. Nevertheless, in a single DBT examination, some organs (such as lung and thyroid) receive higher doses (of about 9% and 21%, respectively) with respect to the CC DM acquisition. CONCLUSIONS: Taking into account an average breast with a thickness of 4.5 cm, the MGDs for DM and DBT acquisitions were below the achievable value (2.0 mGy) defined by the European protocol. Additionally, in the case of a fusion imaging study (DM + DBT), the MGD for a 4.5 cm thick breast is of the order of 1.88 ± 0.36 mGy. Finally, organ dose evaluations underline the need to improve awareness concerning dose estimation of DBT exams for some organs, especially when radiation risk is assessed by using the effective dose.

Are human peripheral nerves sensitive to X-ray imaging?

Diagnostic imaging techniques play an important role in assessing the exact location, cause, and extent of a nerve lesion, thus allowing clinicians to diagnose and manage more effectively a variety of pathological conditions, such as entrapment syndromes, traumatic injuries, and space-occupying lesions. Ultrasound and nuclear magnetic resonance imaging are becoming useful methods for this purpose, but they still lack spatial resolution. In this regard, recent phase contrast x-ray imaging experiments of peripheral nerve allowed the visualization of each nerve fiber surrounded by its myelin sheath as clearly as optical microscopy. In the present study, we attempted to produce high-resolution x-ray phase contrast images of a human sciatic nerve by using synchrotron radiation propagation-based imaging. The images showed high contrast and high spatial resolution, allowing clear identification of each fascicle structure and surrounding connective tissue. The outstanding result is the detection of such structures by phase contrast x-ray tomography of a thick human sciatic nerve section. This may further enable the identification of diverse pathological patterns, such as Wallerian degeneration, hypertrophic neuropathy, inflammatory infiltration, leprosy neuropathy and amyloid deposits. To the best of our knowledge, this is the first successful phase contrast x-ray imaging experiment of a human peripheral nerve sample. Our long-term goal is to develop peripheral nerve imaging methods that could supersede biopsy procedures.

Correlative cryo-fluorescence and cryo-soft X-ray tomography of adherent cells at European synchrotrons.

Cryo-soft X-ray tomography (cryo-SXT) is a synchrotron-hosted imaging technique used to analyze the ultrastructure of intact, cryo-prepared cells. Correlation of cryo-fluorescence microscopy and cryo-SXT can be used to localize fluorescent proteins to organelles preserved close to native state. Cryo-correlative light and X-ray microscopy (cryo-CLXM) is particularly useful for the study of organelles that are susceptible to chemical fixation artifacts during sample preparation for electron microscopy. In our recent work, we used cryo-CLXM to characterize GFP-LC3-positive early autophagosomes in nutrient-starved HEK293A cells (Duke et al., 2013). Cup-shaped omegasomes were found to form at "hot-spots" on the endoplasmic reticulum. Furthermore, cryo-SXT image stacks revealed the presence of large complex networks of tubulated mitochondria in the starved cells, which would be challenging to model at this scale and resolution using light or electron microscopy. In this chapter, we detail the cryo-CLXM workflow that we developed and optimized for studying adherent mammalian cells. We show examples of data collected at the three European synchrotrons that currently host cryo-SXT microscopes, and describe how raw cryo-SXT datasets are processed into tomoX stacks, modeled, and correlated with cryo-fluorescence data to identify structures of interest.

X-ray tomography system to investigate granular materials during mechanical loading.

We integrate a small and portable medical x-ray device with mechanical testing equipment to enable in situ, non-invasive measurements of a granular material's response to mechanical loading. We employ an orthopedic C-arm as the x-ray source and detector to image samples mounted in the materials tester. We discuss the design of a custom rotation stage, which allows for sample rotation and tomographic reconstruction under applied compressive stress. We then discuss the calibration of the system for 3D computed tomography, as well as the subsequent image reconstruction process. Using this system to reconstruct packings of 3D-printed particles, we resolve packing features with 0.52 mm resolution in a (60 mm)(3) field of view. By analyzing the performance bounds of the system, we demonstrate that the reconstructions exhibit only moderate noise.

Improved digital breast tomosynthesis images using automated ultrasound.

PURPOSE: Digital breast tomosynthesis (DBT) offers poor image quality along the depth direction. This paper presents a new method that improves the image quality of DBT considerably through the a priori information from automated ultrasound (AUS) images. METHODS: DBT and AUS images of a complex breast-mimicking phantom are acquired by a DBT/AUS dual-modality system. The AUS images are taken in the same geometry as the DBT images and the gradient information of the in-slice AUS images is adopted into the new loss functional during the DBT reconstruction process. The additional data allow for new iterative equations through solving the optimization problem utilizing the gradient descent method. Both visual comparison and quantitative analysis are employed to evaluate the improvement on DBT images. Normalized line profiles of lesions are obtained to compare the edges of the DBT and AUS-corrected DBT images. Additionally, image quality metrics such as signal difference to noise ratio (SDNR) and artifact spread function (ASF) are calculated to quantify the effectiveness of the proposed method. RESULTS: In traditional DBT image reconstructions, serious artifacts can be found along the depth direction (Z direction), resulting in the blurring of lesion edges in the off-focus planes parallel to the detector. However, by applying the proposed method, the quality of the reconstructed DBT images is greatly improved. Visually, the AUS-corrected DBT images have much clearer borders in both in-focus and off-focus planes, fewer Z direction artifacts and reduced overlapping effect compared to the conventional DBT images. Quantitatively, the corrected DBT images have better ASF, indicating a great reduction in Z direction artifacts as well as better Z resolution. The sharper line profiles along the Y direction show enhancement on the edges. Besides, noise is also reduced, evidenced by the obviously improved SDNR values. CONCLUSIONS: The proposed method provides great improvement on the quality of DBT images. This improvement makes it easier to locate and to distinguish a lesion, which may help improve the accuracy of the diagnosis using DBT imaging.