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Opinion statementProstate cancer (PCa) is the second most diagnosed malignant neoplasm and is one of the leading causes of cancer-related death in men worldwide. Despite significant advances in screening and treatment of PCa, given the... more
Opinion statementProstate cancer (PCa) is the second most diagnosed malignant neoplasm and is one of the leading causes of cancer-related death in men worldwide. Despite significant advances in screening and treatment of PCa, given the heterogeneity of this disease, optimal personalized therapeutic strategies remain limited. However, emerging predictive and prognostic biomarkers based on individual patient profiles in combination with computer-assisted diagnostics have the potential to guide precision medicine, where patients may benefit from therapeutic approaches optimally suited to their disease. Also, the integration of genotypic and phenotypic diagnostic methods is supporting better informed treatment decisions. Focusing on advanced PCa, this review discusses polygenic risk scores for screening of PCa and common genomic aberrations in androgen receptor (AR), PTEN-PI3K-AKT, and DNA damage response (DDR) pathways, considering clinical implications for diagnosis, prognosis, and tr...
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Characterizing the properties of X-ray free-electron laser (XFEL) sources is a critical step for optimization of performance and experiment planning. The recent availability of MHz XFELs has opened up a range of new opportunities for... more
Characterizing the properties of X-ray free-electron laser (XFEL) sources is a critical step for optimization of performance and experiment planning. The recent availability of MHz XFELs has opened up a range of new opportunities for novel experiments but also highlighted the need for systematic measurements of the source properties. Here, MHz-enabled beam imaging diagnostics developed for the SPB/SFX instrument at the European XFEL are exploited to measure the shot-to-shot intensity statistics of X-ray pulses. The ability to record pulse-integrated two-dimensional transverse intensity measurements at multiple planes along an XFEL beamline at MHz rates yields an improved understanding of the shot-to-shot photon beam intensity variations. These variations can play a critical role, for example, in determining the outcome of single-particle imaging experiments and other experiments that are sensitive to the transverse profile of the incident beam. It is observed that shot-to-shot varia...
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Intensity-correlation measurements allow access to nanostructural information on a range of ordered and disordered materials beyond traditional pair-correlation methods. In real space, this information can be expressed in terms of a... more
Intensity-correlation measurements allow access to nanostructural information on a range of ordered and disordered materials beyond traditional pair-correlation methods. In real space, this information can be expressed in terms of a pair-angle distribution function (PADF) which encodes three- and four-body distances and angles. To date, correlation-based techniques have not been applied to the analysis of microstructural effects, such as preferred orientation, which are typically investigated by texture analysis. Preferred orientation is regarded as a potential source of error in intensity-correlation experiments and complicates interpretation of the results. Here, the theory of preferred orientation in intensity-correlation techniques is developed, connecting it to the established theory of texture analysis. The preferred-orientation effect is found to scale with the number of crystalline domains in the beam, surpassing the nanostructural signal when the number of domains becomes l...
Research Interests: Medicine and Correlation
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase (LCP). To date, there has been little discussion in the literature regarding the impact of the... more
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase (LCP). To date, there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase. This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability. Here we report small-angle X-ray scattering measurements of a monoolein:water mixture during continuous delivery using a high viscosity injector. We observe both an alignment and modification of the LCP as a direct result of the injection process. The orientation of the cubic lattice with respect to the beam was estimated based on the anisotropy of the diffraction pattern and does not correspond to a single low order zone axis. The solvent fraction was also observed to impact the stability of the cubic phase during injection. In addition, depending on the distance traveled by the lipid after exiting the needle, the phase is observed to transition from a pure diamond phase (Pn3m) to a mixture containing both gyriod (Ia3d) and lamellar (Lα) phases. Finite element modelling of the observed phase behaviour during injection indicates that the pressure exerted on the lipid stream during extrusion accounts for the variations in the phase composition of the monoolein:water mixture.
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Periodic nanoapertures fabricated in thin metal films exhibit a range of interesting properties in the presence of electromagnetic waves including phenomena such as extraordinary optical transmission (EOT). Fundamentally, these effects... more
Periodic nanoapertures fabricated in thin metal films exhibit a range of interesting properties in the presence of electromagnetic waves including phenomena such as extraordinary optical transmission (EOT). Fundamentally, these effects are mediated by plasmons and have been shown to have a vast range of applications, including, colour filtering, chemical sensing, and as components in solar cells. In the majority of cases, the high spatial resolution required for precise fabrication of these structures is limited to direct writing techniques such as Focused Ion Beam (FIB) and Electron beam lithography (EBL), which only cover relatively small, micron-sized, areas. In this article, we describe and demonstrate the fabrication of plasmonically active devices in the visible range using Displacement Talbot Lithography (DTL). This method allows nanometre-resolution photolithography to be performed over very large areas (whole wafers) without any significant degradation in quality. We presen...
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A serial millisecond crystallography (SMX) facility has recently been implemented at the macromolecular crystallography beamline, MX2 at the Australian Synchrotron. The setup utilizes a combination of an EIGER X 16M detector system and an... more
A serial millisecond crystallography (SMX) facility has recently been implemented at the macromolecular crystallography beamline, MX2 at the Australian Synchrotron. The setup utilizes a combination of an EIGER X 16M detector system and an in-house developed high-viscosity injector, “Lipidico.” Lipidico uses a syringe needle to extrude the microcrystal-containing viscous media and it is compatible with commercially available syringes. The combination of sample delivery via protein crystals suspended in a viscous mixture and a millisecond frame rate detector enables high-throughput serial crystallography at the Australian Synchrotron. A hit-finding algorithm, based on the principles of “robust-statistics,” is employed to rapidly process the data. Here we present the first SMX experimental results with a detector frame rate of 100 Hz (10 ms exposures) and the Lipidico injector using a mixture of lysozyme microcrystals embedded in high vacuum silicon grease. Details of the experimental ...
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The recent development of serial crystallography at synchrotron and X-ray free-electron laser (XFEL) sources is producing crystallographic datasets of ever increasing volume. The size of these datasets is such that fast and efficient... more
The recent development of serial crystallography at synchrotron and X-ray free-electron laser (XFEL) sources is producing crystallographic datasets of ever increasing volume. The size of these datasets is such that fast and efficient analysis presents a range of challenges that have to be overcome to enable real-time data analysis, which is essential for the effective management of XFEL experiments. Among the blocks which constitute the analysis pipeline, one major bottleneck is `peak finding', whose goal is to identify the Bragg peaks within (often) noisy diffraction patterns. Development of faster and more reliable peak-finding algorithms will allow for efficient processing and storage of the incoming data, as well as the optimal use of diffraction data for structure determination. This paper addresses the problem of peak finding and, by extension, `hit finding' in crystallographic XFEL datasets, by exploiting recent developments in robust statistical analysis. The approac...
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Research Interests: Materials Engineering, Mechanical Engineering, Materials Science, Composites, Materials, and 15 moreNanocomposites, Dislocation Dynamics Simulations, Nano, Mechanical properties, Aluminium Alloys, Dependence, Aluminum Alloys, Composite Material, Grain size, Alloy, Metals, Aluminium, Extrusion, Fibre Composites, and Flow Stress
Plasmonic devices provide a unique sensitivity to changes in the permittivity of the immediate, near-surface environment. In this work we explore the use of dual pitch plasmonic devices combined with microfluidics for polarization... more
Plasmonic devices provide a unique sensitivity to changes in the permittivity of the immediate, near-surface environment. In this work we explore the use of dual pitch plasmonic devices combined with microfluidics for polarization enhanced colour sensing of a chemicals’ refractive index. We demonstrate that the use of cross-shaped apertures can produce polarization tunable color based sensing in the optical regime and show that the spectral variations as a function of the incident polarization can be decomposed into contributions from the two orthogonal modes that characterize the dual pitch plasmonic device. Finally we demonstrate that the use of the full colour spectrum in the visible range in combination with polarization control enables sensing ‘by-eye’ of refractive index changes below 1 × 10-3 RIU.
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We present simulations of a square flow focusing droplet generator device exploring its performance characteristics over a range of interfacial surface tension values and varying neck width. Droplet generators have a wide range of... more
We present simulations of a square flow focusing droplet generator device exploring its performance characteristics over a range of interfacial surface tension values and varying neck width. Droplet generators have a wide range of applications from drug delivery to X-ray diffraction experiments. Matching the droplet frequency and volume to the experimental parameters is critical for maximising the data quality and minimising sample waste. Whilst varying the interfacial surface tension we observed that the lowest frequency of droplets is generated for surface tensions matching those typically reported for water-oil mixtures (around 40 mN/M). Decreasing or increasing the interfacial surface tension, for example by adding surfactant, results in an increase in droplet frequency. We also find that under the conditions simulated here, droplets are generated with much lower capillary numbers and higher Weber numbers than have typically been reported in the literature. The high ratio of flo...
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The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates.... more
The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.
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Liquid sample delivery systems are used extensively for serial femtosecond crystallography at X-ray free-electron lasers (XFELs). However, misalignment of the liquid jet and the XFEL beam leads to the X-rays either partially or completely... more
Liquid sample delivery systems are used extensively for serial femtosecond crystallography at X-ray free-electron lasers (XFELs). However, misalignment of the liquid jet and the XFEL beam leads to the X-rays either partially or completely missing the sample, resulting in sample wastage and a loss of experiment time. Implemented here is an algorithm to analyse optical images using machine vision to determine whether there is overlap of the X-ray beam and liquid jet. The long-term goal is to use the output from this algorithm to implement an automated feedback mechanism to maintain constant alignment of the X-ray beam and liquid jet. The key elements of this jet alignment algorithm are discussed and its performance is characterized by comparing the results with a manual analysis of the optical image data. The success rate of the algorithm for correctly identifying hits is quantified via a similarity metric, the Dice coefficient. In total four different nozzle designs were used in this...
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The development of ultrafast X-ray free-electron laser (XFEL) sources and third-generation synchrotrons has opened many new horizons for the study of complex molecular structures and their reaction kinetics. An essential element of these... more
The development of ultrafast X-ray free-electron laser (XFEL) sources and third-generation synchrotrons has opened many new horizons for the study of complex molecular structures and their reaction kinetics. An essential element of these types of experiment is the method used for sample delivery. Microfluidics technology provides the ideal platform for performing these types of measurements since it enables control, manipulation and delivery of small volumes of fluid inside microchannels. Several key functions including mixing, particle separation, and injection, can be integrated on a single chip making the technology very attractive for use in Xray characterisation of molecular dynamics. Key challenges however, in using microfluidics to both mix and deliver samples, include chemical inertness and mechanical stability of the devices, particularly at micron length scales. Here we report a repeatable method for fabricating microfluidic mixer-jet devices based on photolithography and SU8 with a glass substrate. In experiments we have shown that these devices can withstand the high gas pressures required to produce stable, long-range, liquid jets. Coupled with their chemical inertness and reproducibility this makes them promising candidates for time-resolved X-ray diffraction measurements of molecular dynamics. Incorporating an integrated serpentine micromixer capable of homogeneous mixing prior to the liquid jet the devices presented here can be applied to the study of the dynamics of chemically driven biomolecular reactions. The focus of the current work is on the experimental characterization of the mixer through analysis of the concentration profiles along the length of the serpentineshaped microchannel.
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Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of... more
Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 μm x 1.7 μm full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experiment...
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X-ray computed tomography (XCT) is an important clinical diagnostic tool which is also used in a range of biological imaging applications in research. The increasing prevalence of metallic implants in medical and dental radiography and... more
X-ray computed tomography (XCT) is an important clinical diagnostic tool which is also used in a range of biological imaging applications in research. The increasing prevalence of metallic implants in medical and dental radiography and tomography has driven the demand for new approaches to solving the issue of metal artefacts in XCT. Metal artefacts occur when a highly absorbing material is imaged which is in boundary contact with one or more weakly absorbing components, such as soft-tissue. The resulting ‘streaking’ in the reconstructed images creates significant challenges for X-ray analysis due to the non-linear dependence on the absorption properties of the sample. In this paper we introduce a new approach to removing metal artefacts which exploits the capabilities of the recently available, photon-counting PiXirad detector. Our approach works for standard lab-based polychromatic X-ray tubes and does not rely on any postprocessing of the data. The method is demonstrated using bo...
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Ross filter pairs have recently been demonstrated as a highly effective means of producing quasi-monoenergetic beams from polychromatic X-ray sources. They have found applications in both X-ray spectroscopy and for elemental separation in... more
Ross filter pairs have recently been demonstrated as a highly effective means of producing quasi-monoenergetic beams from polychromatic X-ray sources. They have found applications in both X-ray spectroscopy and for elemental separation in X-ray computed tomography (XCT). Here we explore whether they could be applied to the problem of metal artefact reduction (MAR) for applications in medical imaging. Metal artefacts are a common problem in X-ray imaging of metal implants embedded in bone and soft tissue. A number of data post-processing approaches to MAR have been proposed in the literature, however these can be time-consuming and sometimes have limited efficacy. Here we describe and demonstrate an alternative approach based on beam conditioning using Ross filter pairs. This approach obviates the need for any complex post-processing of the data and enables MAR and segmentation from the surrounding tissue by exploiting the absorption edge contrast of the implant.
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Ptychography has recently been adapted for the recovery of the complete Jones matrix of an anisotropic specimen, using a vectorial form of the Ptychographic Iterative Engine (vPIE) for a set of linearly polarized probes. Here we show that... more
Ptychography has recently been adapted for the recovery of the complete Jones matrix of an anisotropic specimen, using a vectorial form of the Ptychographic Iterative Engine (vPIE) for a set of linearly polarized probes. Here we show that this method can be applied to the recovery of the in-plane components of the elastic strain tensor in a diametrically compressed disc. The advantages and disadvantages of vPIE for the recovery of strain information from ‘real-world’ samples is discussed as well as the potential for this approach to be applied to the characterization of the mechanical properties of optically transparent materials
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Determining the specific spatial distributions of elements within compound samples is of critical importance to a range of applied research fields. The usual approaches to obtaining elemental contrast involve measurement of the... more
Determining the specific spatial distributions of elements within compound samples is of critical importance to a range of applied research fields. The usual approaches to obtaining elemental contrast involve measurement of the characteristic peaks associated with x-ray fluorescence or measuring the x-ray transmission as a function of energy. In the laboratory these measurements are challenging due to the polychromaticity and lack of tunability of the source. Here we demonstrate how newly developed, high-resolution, energy-discriminating area detector technology can be exploited to enhance elemental contrast. The detector we employ here is the Pixirad area detector which can simultaneously have up to four separate colour channels. We also discuss the potential of this new technology in the context of tomographic imaging of soft tissue.
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Three-dimensional imaging of protein crystals during x-ray diffraction experiments opens up a range of possibilities for optimizing crystal quality and gaining new insights into the fundamental processes that drive radiation damage.... more
Three-dimensional imaging of protein crystals during x-ray diffraction experiments opens up a range of possibilities for optimizing crystal quality and gaining new insights into the fundamental processes that drive radiation damage. Obtaining this information at the appropriate length-scales however is extremely challenging. One approach that has been recently demonstrated as a promising avenue for characterizing the size and shape of protein crystals at nanometre length-scales is Bragg coherent diffractive imaging (BCDI). BCDI is a recently developed technique that is able to recover the phase of the continuous diffraction intensity signal around individual Bragg peaks. When data is collected at multiple points on a rocking curve, a reciprocal space map (RSM) can be assembled and then inverted using BCDI to obtain a three-dimensional image of the crystal. The first demonstration of two-dimensional biological BCDI was reported by Boutet et al on holoferritin, recently this work was extended to the study of radiation damage in micron-sized protein crystals. Here we present the first three-dimensional reconstructions of a Lysozyme protein crystal using BDI. The results are validated against RSM and transmission electron microscopy data and have implications for both radiation damage studies and for developing new approaches for structure retrieval from micron-sized protein crystals.
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We present single-shot images of a laser-driven shock wave interacting with micron-scale materials heterogeneities taken with an X-ray free electron laser. We observed an evolution from an elastic to plastic shock wave in uniaxially... more
We present single-shot images of a laser-driven shock wave interacting with micron-scale materials heterogeneities taken with an X-ray free electron laser. We observed an evolution from an elastic to plastic shock wave in uniaxially loaded LiF and PETN.
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Periodic subwavelength apertures have the ability to passively detect variations in the dielectric properties of the local sample environment through modification of the plasmon resonances associated with these structures. The resulting... more
Periodic subwavelength apertures have the ability to passively detect variations in the dielectric properties of the local sample environment through modification of the plasmon resonances associated with these structures. The resulting resonance peak can effectively provide a ‘fingerprint’ indicative of the dielectric properties of the medium within the near-surface region. Here we report on the use of biomodal silver-based plasmonic colour filters for molecular sensing. Firstly, by characterising the optical properties of these devices as a function of the incident polarisation for a range of different analytes of known refractive index (RI), we were able to both maximise and quantify their sensitivity. We then apply this concept to the real-time monitoring of the formation of self-assembled monolayers (SAMs) based on detection of the optical output using a spectrometer. This highlights the potential for bimodal plasmonic devices to be able to dynamically monitor variations in the...
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Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used... more
Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The r...
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Fluctuation x-ray scattering data collected at the Small-angle x-ray scattering (SAXS) beamline at the Australian Synchrotron. Samples are self-assembled Monoolein:buffer mixtures with additives: cholesterol and... more
Fluctuation x-ray scattering data collected at the Small-angle x-ray scattering (SAXS) beamline at the Australian Synchrotron. Samples are self-assembled Monoolein:buffer mixtures with additives: cholesterol and 1,2-Dioleoyl-phosphatidylcholine (DOPC) in varying concentrations. Please see the manuscript for composition of the buffer solution. Pn3m and hexagonal phases were observed. Around ~3000 diffraction patterns of independent sample regions were collected for each sample composition, in order to study angular intensity correlations.<br>The file names convention is as follows:"run8_3p5dopc_3p7chol.tar.gz" indicates a MO:buffer mixture for 3.5% DOPC and 3.7% cholesterol.<br>See the following article for more information:Martin, A.V., Kozlov, A., Berntsen, P. et al. Fluctuation X-ray diffraction reveals three-dimensional nanostructure and disorder in self-assembled lipid phases. Commun Mater 1, 40 (2020). https://doi.org/10.1038/s43246-020-0044-z<br>
X-ray Free-Electron Lasers (XFELs) offer a unique opportunity to study the structural dynamics of proteins on a femtosecond time-scale. To realize the full potential of XFEL sources for studying time-resolved biomolecular processes... more
X-ray Free-Electron Lasers (XFELs) offer a unique opportunity to study the structural dynamics of proteins on a femtosecond time-scale. To realize the full potential of XFEL sources for studying time-resolved biomolecular processes however, requires the optimization and development of devices that can both act as a trigger and a delivery mechanism for the system of interest. Here we present numerical simulations and actual devices exploring the conditions required for the development of successful mixing and injection devices for tracking the molecular dynamics of proteins in solution on micro to nanosecond timescales using XFELs. The mechanism for combining reagents employs a threefold combination of pico-liter volumes, lamination and serpentine mixing. Focusing and delivering the sample in solution is achieved using the Gas Dynamic Virtual Nozzle (GDVN), which was specifically developed to produce a micrometer diameter, in-vacuum liquid jet. We explore the influence of parameters such as flow rate and gas pressure on the mixing time and jet stability, and explore the formation of rapid homogeneously mixed jets for ‘mix-and-inject’ liquid scattering experiments at Synchrotron and XFEL facilities.
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Synchrotron-based micro-beam Laue diffraction is an experimental technique for the study of intra-granular lattice orientation and elastic strain in individual crystallites of polycrystalline engineering alloys. Traditionally the... more
Synchrotron-based micro-beam Laue diffraction is an experimental technique for the study of intra-granular lattice orientation and elastic strain in individual crystallites of polycrystalline engineering alloys. Traditionally the technique operates in reflection geometry with a polychromatic X-ray beam focused to a sub-micron spot and with photon energy ranging from approximately 5 to 30keV. This allows the study of material in the near-surface region. In this paper the first feasibility study of extending this technique to a ...
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Non-destructive, quantitative and precise determination of internal strain distributions within structural materials and components can be accomplished by only a few experimental techniques, amongst which diffraction of penetrating... more
Non-destructive, quantitative and precise determination of internal strain distributions within structural materials and components can be accomplished by only a few experimental techniques, amongst which diffraction of penetrating radiation (X-ray and neutron beams) plays a central role. However, due to the various limitations of these methods, the 2-D and 3-D mapping of internal strains within bulk cross-sections at a spatial resolution of 0.1 mm or better has long remained a challenge. The principle of ���strain tomography��� proposed by ...
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The Imaging and Medical Beamline (IMBL) is a superconducting multipole wiggler-based beamline at the 3 GeV Australian Synchrotron operated by the Australian Nuclear Science and Technology Organisation (ANSTO). The beamline delivers hard... more
The Imaging and Medical Beamline (IMBL) is a superconducting multipole wiggler-based beamline at the 3 GeV Australian Synchrotron operated by the Australian Nuclear Science and Technology Organisation (ANSTO). The beamline delivers hard X-rays in the 25–120 keV energy range and offers the potential for a range of biomedical X-ray applications, including radiotherapy and medical imaging experiments. One of the imaging modalities available at IMBL is propagation-based X-ray phase-contrast computed tomography (PCT). PCT produces superior results when imaging low-density materials such as soft tissue (e.g., breast mastectomies) and has the potential to be developed into a valuable medical imaging tool. We anticipate that PCT will be utilized for medical breast imaging in the near future with the advantage that it could provide better contrast than conventional X-ray absorption imaging. The unique properties of synchrotron X-ray sources such as high coherence, energy tunability, and high...
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The majority of lab-based X-ray sources are polychromatic and are not easily tunable, which can make the 3D quantitative analysis of multi-component samples challenging. The lack of effective materials separation when using conventional... more
The majority of lab-based X-ray sources are polychromatic and are not easily tunable, which can make the 3D quantitative analysis of multi-component samples challenging. The lack of effective materials separation when using conventional X-ray tube sources has motivated the development of a number of potential solutions including the application of dual-energy X-ray computed tomography (CT) as well as the use of X-ray filters. Here, we demonstrate the simultaneous decomposition of two low-density materials via inversion of the linear attenuation matrices using data from the energy-discriminating PiXirad detector. A key application for this method is soft-tissue differentiation which is widely used in biological and medical imaging. We assess the effectiveness of this approach using both simulation and experiment noting that none of the materials investigated here incorporate any contrast enhancing agents. By exploiting the energy discriminating properties of the detector, narrow ener...
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This journal is © The Royal Society of Chemistry. Serial femtosecond crystallography (SFX) methods used at X-ray free electron lasers (XFELs) offer a range of new opportunities for structural biology. A crucial component of SFX... more
This journal is © The Royal Society of Chemistry. Serial femtosecond crystallography (SFX) methods used at X-ray free electron lasers (XFELs) offer a range of new opportunities for structural biology. A crucial component of SFX experiments is sample delivery. Microfluidic devices can be employed in SFX experiments to precisely deliver microcrystals to the X-ray beam and to trigger molecular dynamics via rapid mix-and-inject measurements. Here, for the first time, we have developed a process based on high-resolution photolithography using SU8 on glass to fabricate microfluidic mix-and-inject devices. In order to characterise these devices a broad range of flow rates are used and the mixing and jetting response of the devices monitored. We observe that a stable jet is formed using these devices when injecting DI-water. Three different jetting regimes, liquid column, ribbon, and cylindrical jet, were observed. Furthermore, fluorescence experiments confirm that rapid and uniform mixing ...
Serial Synchrotron Crystallography (SSX) is rapidly emerging as a promising technique for collecting data for time-resolved structural studies or for performing room temperature micro-crystallography measurements using micro-focused... more
Serial Synchrotron Crystallography (SSX) is rapidly emerging as a promising technique for collecting data for time-resolved structural studies or for performing room temperature micro-crystallography measurements using micro-focused beamlines. SSX is often performed using high frame rate detectors in combination with continuous sample scanning or high-viscosity or liquid jet injectors. When performed using ultra-bright X-ray Free Electron Laser (XFEL) sources serial crystallography typically involves a process known as ’diffract-and-destroy’ where each crystal is measured just once before it is destroyed by the intense XFEL pulse. In SSX, however, particularly when using high-viscosity injectors (HVIs) such as Lipidico, the crystal can be intercepted multiple times by the X-ray beam prior to exiting the interaction region. This has a number of important consequences for SSX including whether these multiple-hits can be incorporated into the data analysis or whether they need to be ex...
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Microfluidic devices which integrate both rapid mixing and liquid jetting for sample delivery are an emerging solution for studying molecular dynamics via X-ray diffraction. Here we use finite element modelling to investigate the... more
Microfluidic devices which integrate both rapid mixing and liquid jetting for sample delivery are an emerging solution for studying molecular dynamics via X-ray diffraction. Here we use finite element modelling to investigate the efficiency and time-resolution achievable using microfluidic mixers within the parameter range required for producing stable liquid jets. Three-dimensional simulations, validated by experimental data, are used to determine the velocity and concentration distribution within these devices. The results show that by adopting a serpentine geometry, it is possible to induce chaotic mixing, which effectively reduces the time required to achieve a homogeneous mixture for sample delivery. Further, we investigate the effect of flow rate and the mixer microchannel size on the mixing efficiency and minimum time required for complete mixing of the two solutions whilst maintaining a stable jet. In general, we find that the smaller the cross-sectional area of the mixer mi...
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Microfluidic devices which integrate both rapid mixing and liquid jetting for sample delivery are an emerging solution for studying molecular dynamics via X-ray diffraction. Here we use finite element modelling to investigate the... more
Microfluidic devices which integrate both rapid mixing and liquid jetting for sample delivery are an emerging solution for studying molecular dynamics via X-ray diffraction. Here we use finite element modelling to investigate the efficiency and time-resolution achievable using microfluidic mixers within the parameter range required for producing stable liquid jets. Three-dimensional simulations, validated by experimental data, are used to determine the velocity and concentration distribution within these devices. The results show that by adopting a serpentine geometry, it is possible to induce chaotic mixing, which effectively reduces the time required to achieve a homogeneous mixture for sample delivery. Further, we investigate the effect of flow rate and the mixer microchannel size on the mixing efficiency and minimum time required for complete mixing of the two solutions whilst maintaining a stable jet. In general, we find that the smaller the cross-sectional area of the mixer mi...
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A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography... more
A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to >10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.
MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline... more
MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct compa...
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The invention of phase contrast microscopy revolutionized optics, enabling the visualization of highly optically transparent samples without the need for staining. The technique utilizes phase shifts within the sample and is routinely... more
The invention of phase contrast microscopy revolutionized optics, enabling the visualization of highly optically transparent samples without the need for staining. The technique utilizes phase shifts within the sample and is routinely employed in the characterization of biological material and other weakly interacting objects. However, the demand for increased contrast and quantification has continued to drive research into more advanced approaches to phase imaging. Here, we demonstrate that the combination of ptychographic coherent diffractive imaging with plasmonically active metamaterials yields a massive enhancement of both the reconstructed phase and amplitude by exploiting near-field interactions at the metamaterial surface. We present results from nanofabricated samples and tissue sections with thickness ranging from 4 nm to 4 μm. In addition to enabling quantitative phase imaging of metamaterials, this approach opens the way to imaging a wide range of extremely thin or highly transparent objects previously inaccessible to optical microscopy. Plasmonics and metamaterials enable ptychographic coherent diffractive imaging with improved reconstructed phase and amplitude. The approach may be particularly useful for imaging of extremely thin or highly transparent objects.