Shielding design aspects of SR in 3 GeV ILSF.

Iranian Light Source Facility (ILSF) is an under-construction synchrotron radiation accelerator consisting of a 150 MeV linac, a booster synchrotron operating from 150 MeV to 3 GeV, and a 3 GeV storage ring that stores a maximum of 400 mA current of electrons. As the stored beam circulates, a fraction of the beam is lost due to interactions with gas molecules, interactions among beam particles, and orbital bending, which produce radiation. The bulk shielding calculation for the ILSF and the input parameters used for this analysis are discussed in this paper. The potential of skyshine neutrons to cause radiation hazards is investigated as well. Moreover, the design and shielding simulation using the FLUKA Monte Carlo code is presented for the linac beam stop and primary and scattered gas bremsstrahlung for the first optics enclosure of the ILSF spectro microscopy beamline. Our designed radiation shielding system guarantees that the annual dose in all areas around the ILSF machine does not exceed the dose limit of 1 mSv.

In-Situ Synchrotron SAXS and WAXS Investigation on the Deformation of Single and Coaxial Electrospun P(VDF-TrFE)-Based Nanofibers.

Coaxial core/shell electrospun nanofibers consisting of ferroelectric P(VDF-TrFE) and relaxor ferroelectric P(VDF-TrFE-CTFE) are tailor-made with hierarchical structures to modulate their mechanical properties with respect to their constituents. Compared with two single and the other coaxial membranes prepared in the research, the core/shell-TrFE/CTFE membrane shows a more prominent mechanical anisotropy between revolving direction (RD) and cross direction (CD) associated with improved resistance to tensile stress for the crystallite phase stability and good strength-ductility balance. This is due to the better degree of core/shell-TrFE-CTFE nanofiber alignment and the crystalline/amorphous ratio. The coupling between terpolymer P(VDF-TrFE-CTFE) and copolymer P(VDF-TrFE) is responsible for phase stabilization, comparing the core/shell-TrFE/CTFE with the pristine terpolymer. Moreover, an impressive collective deformation mechanism of a two-length scale in the core/shell composite structure is found. We apply in-situ synchrotron X-ray to resolve the two-length scale simultaneously by using the small-angle X-ray scattering to characterize the nanofibers and the wide-angle X-ray diffraction to identify the phase transformations. Our findings may serve as guidelines for the fabrication of the electrospun nanofibers used as membranes-based electroactive polymers.

No Intercellular Regulation of the Cell Cycle among Human Cervical Carcinoma HeLa Cells Expressing Fluorescent Ubiquitination-Based Cell-Cycle Indicators in Modulated Radiation Fields.

The non-targeted effects of radiation have been known to induce significant alternations in cell survival. Although the effects might govern the progression of tumor sites following advanced radiotherapy, the impacts on the intercellular control of the cell cycle following radiation exposure with a modified field, remain to be determined. Recently, a fluorescent ubiquitination-based cell-cycle indicator (FUCCI), which can visualize the cell-cycle phases with fluorescence microscopy in real time, was developed for biological cell research. In this study, we investigated the non-targeted effects on the regulation of the cell cycle of human cervical carcinoma (HeLa) cells with imperfect p53 function that express the FUCCI (HeLa-FUCCI cells). The possible effects on the cell-cycle phases via soluble factors were analyzed following exposure to different field configurations, which were delivered using a 150 kVp X-ray irradiator. In addition, using synchrotron-generated, 5.35 keV monochromatic X-ray microbeams, high-precision 200 µm-slit microbeam irradiation was performed to investigate the possible impacts on the cell-cycle phases via cell-cell contacts. Collectively, we could not detect the intercellular regulation of the cell cycle in HeLa-FUCCI cells, which suggested that the unregulated cell growth was a malignant tumor. Our findings indicated that there was no significant intercellular control system of the cell cycle in malignant tumors during or after radiotherapy, highlighting the differences between normal tissue and tumor characteristics.

Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase.

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5-15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3-5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Šin SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Šresolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

Label-Free, Real-Time Measurement of Metabolism of Adherent and Suspended Single Cells by In-Cell Fourier Transform Infrared Microspectroscopy.

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.

Novel combined crystallization plate for high-throughput crystal screening and in situ data collection at a crystallography beamline.

In situ microplates are small in size, crystal cultivation and operation are difficult, and the efficiency of crystal screening is relatively low. To solve this problem, a novel combined crystallization plate was designed for high-throughput crystal cultivation and in situ data collection. A frame was used to hold 48 in situ microplates, and the in situ microplates were sealed on one side with an ultralow background-scattering Kapton film. An automatic liquid handler (Mosquito) was used to add a liquid drop to the in situ microplates in the frame, and CrystalClear HD tape was used to seal the frame. A sealed frame holding 48 microplates was developed as a novel combined crystallization plate and was used for crystal cultivation under different conditions and in situ data collection at the synchrotron beamline. Moreover, individual microplates can be separated from the combined crystal plate and then fixed on a magnetic base or loaded onto a UniPuck for in situ data collection. Automatic grid scanning was used to locate crystals. The efficiency of the combined crystallization plate for crystal screening was verified. This method avoids the manual manipulation of crystals during crystal screening and diffraction data collection; therefore, the combined crystallization plate is suitable for large-scale screening of microcrystals.

New high-throughput endstation to accelerate the experimental optimization pipeline for synchrotron X-ray footprinting.

Synchrotron X-ray footprinting (XF) is a growing structural biology technique that leverages radiation-induced chemical modifications via X-ray radiolysis of water to produce hydroxyl radicals that probe changes in macromolecular structure and dynamics in solution states of interest. The X-ray Footprinting of Biological Materials (XFP) beamline at the National Synchrotron Light Source II provides the structural biology community with access to instrumentation and expert support in the XF method, and is also a platform for development of new technological capabilities in this field. The design and implementation of a new high-throughput endstation device based around use of a 96-well PCR plate form factor and supporting diagnostic instrumentation for synchrotron XF is described. This development enables a pipeline for rapid comprehensive screening of the influence of sample chemistry on hydroxyl radical dose using a convenient fluorescent assay, illustrated here with a study of 26 organic compounds. The new high-throughput endstation device and sample evaluation pipeline now available at the XFP beamline provide the worldwide structural biology community with a robust resource for carrying out well optimized synchrotron XF studies of challenging biological systems with complex sample compositions.

Tools for supporting solution scattering during the COVID-19 pandemic.

During the COVID-19 pandemic, synchrotron beamlines were forced to limit user access. Performing routine measurements became a challenge. At the Life Science X-ray Scattering (LiX) beamline, new instrumentation and mail-in protocols have been developed to remove the access barrier to solution scattering measurements. Our efforts took advantage of existing instrumentation and coincided with the larger effort at NSLS-II to support remote measurements. Given the limited staff-user interaction for mail-in measurements, additional software tools have been developed to ensure data quality, to automate the adjustments in data processing, as users would otherwise rely on the experience of the beamline staff, and produce a summary of the initial assessments of the data. This report describes the details of these developments.

Study of the Synchrotron Photoionization Oxidation of Alpha-Angelica Lactone (AAL) Initiated by O(3P) at 298, 550, and 700 K.

In recent years, biofuels have been receiving significant attention because of their potential for decreasing carbon emissions and providing a long-term renewable solution to unsustainable fossil fuels. Currently, lactones are some of the alternatives being produced. Many lactones occur in a range of natural substances and have many advantages over bioethanol. In this study, the oxidation of alpha-angelica lactone initiated by ground-state atomic oxygen, O(3P), was studied at 298, 550, and 700 K using synchrotron radiation coupled with multiplexed photoionization mass spectrometry at the Lawrence Berkeley National Lab (LBNL). Photoionization spectra and kinetic time traces were measured to identify the primary products. Ketene, acetaldehyde, methyl vinyl ketone, methylglyoxal, dimethyl glyoxal, and 5-methyl-2,4-furandione were characterized as major reaction products, with ketene being the most abundant at all three temperatures. Possible reaction pathways for the formation of the observed primary products were computed using the CBS-QB3 composite method.

Integrated sample-handling and mounting system for fixed-target serial synchrotron crystallography.

Serial synchrotron crystallography (SSX) is enabling the efficient use of small crystals for structure-function studies of biomolecules and for drug discovery. An integrated SSX system has been developed comprising ultralow background-scatter sample holders suitable for room and cryogenic temperature crystallographic data collection, a sample-loading station and a humid `gloveless' glovebox. The sample holders incorporate thin-film supports with a variety of designs optimized for different crystal-loading challenges. These holders facilitate the dispersion of crystals and the removal of excess liquid, can be cooled at extremely high rates, generate little background scatter, allow data collection over >90° of oscillation without obstruction or the risk of generating saturating Bragg peaks, are compatible with existing infrastructure for high-throughput cryocrystallography and are reusable. The sample-loading station allows sample preparation and loading onto the support film, the application of time-varying suction for optimal removal of excess liquid, crystal repositioning and cryoprotection, and the application of sealing films for room-temperature data collection, all in a controlled-humidity environment. The humid glovebox allows microscope observation of the sample-loading station and crystallization trays while maintaining near-saturating humidities that further minimize the risks of sample dehydration and damage, and maximize working times. This integrated system addresses common problems in obtaining properly dispersed, properly hydrated and isomorphous microcrystals for fixed-orientation and oscillation data collection. Its ease of use, flexibility and optimized performance make it attractive not just for SSX but also for single-crystal and few-crystal data collection. Fundamental concepts that are important in achieving desired crystal distributions on a sample holder via time-varying suction-induced liquid flows are also discussed.

Distinct types of plexiform lesions identified by synchrotron-based phase-contrast micro-CT.

In pulmonary arterial hypertension, plexiform lesions are associated with severe arterial obstruction and right ventricular failure. Exploring their structure and position is crucial for understanding the interplay between hemodynamics and vascular remodeling. The aim of this research was to use synchrotron-based phase-contrast micro-CT to study the three-dimensional structure of plexiform lesions. Archived paraffin-embedded tissue samples from 14 patients with pulmonary arterial hypertension (13 idiopathic, 1 with known BMPR2-mutation) were imaged. Clinical data showed high-median PVR (12.5 WU) and mPAP (68 mmHg). Vascular lesions with more than 1 lumen were defined as plexiform. Prior radiopaque dye injection in some samples facilitated 3-D rendering. Four distinct types of plexiform lesions were identified: 1) localized within or derived from monopodial branches (supernumerary arteries), often with a connection to the vasa vasorum; 2) localized between pulmonary arteries and larger airways as a tortuous transformation of intrapulmonary bronchopulmonary anastomoses; 3) as spherical structures at unexpected abrupt ends of distal pulmonary arteries; and 4) as occluded pulmonary arteries with recanalization. By appearance and localization, types 1-2 potentially relieve pressure via the bronchial circulation, as pulmonary arteries in these patients were almost invariably occluded distally. In addition, types 1-3 were often surrounded by dilated thin-walled vessels, often connected to pulmonary veins, peribronchial vessels, or the vasa vasorum. Collaterals, bypassing completely occluded pulmonary arteries, were also observed to originate within plexiform lesions. In conclusion, synchrotron-based imaging revealed significant plexiform lesion heterogeneity, resulting in a novel classification. The four types likely have different effects on hemodynamics and disease progression.

Synchrotron micro-X-ray fluorescence imaging of arsenic in frozen-hydrated sections of a root of Pteris vittata.

We performed micro-X-ray fluorescence imaging of frozen-hydrated sections of a root of Pteris vittata for the first time, to the best of our knowledge, to reveal the mechanism of arsenic (As) uptake. The As distribution was successfully visualized in cross sections of different parts of the root, which showed that (i) the major pathway of As uptake changes from symplastic to apoplastic transport in the direction of root growth, and (ii) As and K have different mobilities around the stele before xylem loading, despite their similar distributions outside the stele in the cross sections. These data can reasonably explain As reduction, axially observed around the root tip in the direction of root growth and radially observed in the endodermis in the cross sections, as a consequence of the incorporation of As into the cells or symplast of the root. In addition, previous observations of As species in the midrib can be reconciled by ascribing a reduction capacity to the root cells, which implies that As reduction mechanisms at the cellular level may be an important control on the peculiar root-to-shoot transport of As in P. vittata.

Tracking biochemical changes induced by iron loading in AML12 cells with synchrotron live cell, time-lapse infrared microscopy.

Hepatocytes are essential for maintaining the homeostasis of iron and lipid metabolism in mammals. Dysregulation of either iron or lipids has been linked with serious health consequences, including non-alcoholic fatty liver disease (NAFLD). Considered the hepatic manifestation of metabolic syndrome, NAFLD is characterised by dysregulated lipid metabolism leading to a lipid storage phenotype. Mild to moderate increases in hepatic iron have been observed in ∼30% of individuals with NAFLD; however, direct observation of the mechanism behind this increase has remained elusive. To address this issue, we sought to determine the metabolic consequences of iron loading on cellular metabolism using live cell, time-lapse Fourier transform infrared (FTIR) microscopy utilising a synchrotron radiation source to track biochemical changes. The use of synchrotron FTIR is non-destructive and label-free, and allowed observation of spatially resolved, sub-cellular biochemical changes over a period of 8 h. Using this approach, we have demonstrated that iron loading in AML12 cells induced perturbation of lipid metabolism congruent with steatosis development. Iron-loaded cells had approximately three times higher relative ester carbonyl concentration compared with controls, indicating an accumulation of triglycerides. The methylene/methyl ratio qualitatively suggests the acyl chain length of fatty acids in iron-loaded cells increased over the 8 h period of monitoring compared with a reduction observed in the control cells. Our findings provide direct evidence that mild to moderate iron loading in hepatocytes drives de novo lipid synthesis, consistent with a role for iron in the initial hepatic lipid accumulation that leads to the development of hepatic steatosis.

Proton FLASH: passive scattering or pencil beam scanning?

This study focused on a direct comparison of dose delivery efficiency between two proton FLASH delivery modes: passive scattering and pencil beam scanning (PBS). Monte-Carlo simulation of the beamline was performed using the Geant4 package. Two proton energies (63 and 230 MeV) were selected, targeting for shallow and deep-seated tumors, respectively. Two irradiation field sizes were selected: 13 × 13 mm2 and 50 × 50 mm2. For each delivery mode, two cases were investigated: shoot-through and Bragg peak, yielding a total of 4 delivery scenarios. For the passive scattering mode, the impact on dose rate by multiple components along the beamline were investigated, including ridge-filter, scatterer, range shifter and collimator. A quantitative comparison among four scenarios was made in terms of field size, dose, dose rate and treatment plan quality (dose volume histogram). For the 230 MeV case, the dose rate (for 1 nA current) is 0.05 Gy s-1 (passive with Bragg peak, field size: 50 × 50 mm2) and 2.6 Gy s-1 (PBS with shoot-through). Dose rate comparison is made between passive scattering and PBS as the delivery changes from spot-layer to shoot-through. In conclusion, the study successfully established a benchmark reference for dose rate performance for different scenarios, taking into account components along the beamline, field size and beam current. The results allow us to predict and compare the required beam current to yield a dose rate sufficiently high, above the threshold of the FLASH effect.

A commercial treatment planning system with a hybrid dose calculation algorithm for synchrotron radiotherapy trials.

Synchrotron Radiotherapy (SyncRT) is a preclinical radiation treatment which delivers synchrotron x-rays to cancer targets. SyncRT allows for novel treatments such as Microbeam Radiotherapy, which has been shown to have exceptional healthy tissue sparing capabilities while maintaining good tumour control. Veterinary trials in SyncRT are anticipated to take place in the near future at the Australian Synchrotron's Imaging and Medical Beamline (IMBL). However, before veterinary trials can commence, a computerised treatment planning system (TPS) is required, which can quickly and accurately calculate the synchrotron x-ray dose through patient CT images. Furthermore, SyncRT TPS's must be familiar and intuitive to radiotherapy planners in order to alleviate necessary training and reduce user error. We have paired an accurate and fast Monte Carlo (MC) based SyncRT dose calculation algorithm with EclipseTM, the most widely implemented commercial TPS in the clinic. Using EclipseTM, we have performed preliminary SyncRT trials on dog cadavers at the IMBL, and verified calculated doses against dosimetric measurement to within 5% for heterogeneous tissue-equivalent phantoms. We have also performed a validation of the TPS against a full MC simulation for constructed heterogeneous phantoms in EclipseTM, and showed good agreement for a range of water-like tissues to within 5%-8%. Our custom EclipseTM TPS for SyncRT is ready to perform live veterinary trials at the IMBL.

A new generation of direct X-ray detectors for medical and synchrotron imaging applications.

Large-area X-ray imaging is one of the most widely used imaging modalities that spans several scientific and technological fields. Currently, the direct X-ray conversion materials that are being commercially used for large-area (> 8 cm × 4 cm without tiling) flat panel applications, such as amorphous selenium (a-Se), have usable sensitivities of up to only 30 keV. Although there have been many promising candidates (such as polycrystalline HgI2 and CdTe), none of the semiconductors were able to assuage the requirement for high energy (> 40 keV) large-area X-ray imaging applications due to inadequate cost, manufacturability, and long-term performance metrics. In this study, we successfully demonstrate the potential of the hybrid Methylammonium lead iodide (MAPbI3) perovskite-based semiconductor detectors in satisfying all the requirements for its successful commercialization in synchrotron and medical imaging. This new generation of hybrid detectors demonstrates low dark current under electric fields needed for high sensitivity X-ray imaging applications. The detectors have a linear response to X-ray energy and applied bias, no polarization effects at a moderate bias, and signal stability over long usage durations. Also, these detectors have demonstrated a stable detection response under BNL's National Synchrotron Light Source II (NSLS-II) 70 keV monochromatic synchrotron beamline.

High-throughput in situ experimental phasing.

In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.

Synchrotron FTIR microspectroscopy revealed apoptosis-induced biomolecular changes of cholangiocarcinoma cells treated with ursolic acid.

BACKGROUND: Ursolic acid (UA) is a natural triterpenoid which possesses anti-cancer activity. However, little is known regarding the activity and molecular mechanism of UA in cholangiocarcinoma (CCA). Thus, we investigated the effects of UA on growth inhibition and apoptosis induction through biomolecular changes in KKU-213 and KKU-055 CCA cell lines. METHODS: The anti-proliferative effect of UA against CCA cells was evaluated using SRB assay. Changes in biomolecules were assessed by SR-FTIR microspectroscopy combined with PCA and conventional methods (i.e., Annexin V-FITC/PI staining for lipid alteration and apoptosis induction; Western blot analysis and caspase-3/7 activity assay for apoptotic protein detection). RESULTS: UA suppressed the proliferation of CCA cells in a dose- and time-dependent manner. SR-FTIR data revealed a significant alteration in lipids attributable to changes in apoptotic cell membranes, confirmed by Annexin V-FITC/PI staining. SR-FTIR data showed that UA promoted changes in the protein secondary structure. Elevated expression of Bax and decreased expression of Bcl-2 and survivin/BIRC5 along with augmented caspase-3/7 activity supported alterations in apoptosis-related proteins. CONCLUSIONS: SR-FTIR microspectroscopy was successfully used as a label-free technique to monitor apoptosis-induced biomolecular changes in UA-treated CCA cells. UA exerted the cytotoxic and apoptotic activities in CCA cells through alterations in membrane lipids and apoptotic proteins. UA could be a potential anti-CCA candidate and a chemical starting point for the discovery of novel anti-cancer agents. SIGNIFICANCE: Our present study showed the first evidence that UA exhibited the anti-proliferative and pro-apoptotic activities toward CCA cells through changes in biomolecules, notably lipids and proteins.

Semiconductor dosimetry in modern external-beam radiation therapy.

Semiconductor dosimeters are ubiquitous in modern external-beam radiation therapy. They possess key features. The response, electronically available in real time, is stable and linear with absorbed dose for given irradiation conditions; the radiation-sensitive volume can be rather small in size, while retaining mechanical strength and high sensitivity. We describe three common semiconductor dosimeters: diodes, metal-oxide-semiconductor field-effect transistors and diamonds. We discuss in detail their operation principles and applications in modern external-beam radiation therapy, primarily with megavoltage photon beams. We also explore their use in proton and heavy ion therapy, and in experimental radiotherapy techniques such as synchrotron-based micro-beam radiation therapy.

Nano-imaging trace elements at organelle levels in substantia nigra overexpressing α-synuclein to model Parkinson's disease.

Sub-cellular trace element quantifications of nano-heterogeneities in brain tissues offer unprecedented ways to explore at elemental level the interplay between cellular compartments in neurodegenerative pathologies. We designed a quasi-correlative method for analytical nanoimaging of the substantia nigra, based on transmission electron microscopy and synchrotron X-ray fluorescence. It combines ultrastructural identifications of cellular compartments and trace element nanoimaging near detection limits, for increased signal-to-noise ratios. Elemental composition of different organelles is compared to cytoplasmic and nuclear compartments in dopaminergic neurons of rat substantia nigra. They exhibit 150-460 ppm of Fe, with P/Zn/Fe-rich nucleoli in a P/S-depleted nuclear matrix and Ca-rich rough endoplasmic reticula. Cytoplasm analysis displays sub-micron Fe/S-rich granules, including lipofuscin. Following AAV-mediated overexpression of α-synuclein protein associated with Parkinson's disease, these granules shift towards higher Fe concentrations. This effect advocates for metal (Fe) dyshomeostasis in discrete cytoplasmic regions, illustrating the use of this method to explore neuronal dysfunction in brain diseases.