A Photodynamic and Photochemotherapeutic Platinum-Iridium Charge-Transfer Conjugate for Anticancer Therapy.

The novel hetero-dinuclear complex trans,trans,trans-[PtIV(py)2(N3)2(OH)(µ-OOCCH2CH2CONHCH2-bpyMe)IrIII(ppy)2]Cl (Pt-Ir), exhibits charge transfer between the acceptor photochemotherapeutic Pt(IV) (Pt-OH) and donor photodynamic Ir(III) (Ir-NH2) fragments. It is stable in the dark, but undergoes photodecomposition more rapidly than the Pt(IV) parent complex (Pt-OH) to generate Pt(II) species, an azidyl radical and 1O2. The Ir(III)* excited state, formed after irradiation, can oxidise NADH to NAD⋅ radicals and NAD+. Pt-Ir is highly photocytotoxic towards cancer cells with a high photocytotoxicity index upon irradiation with blue light (465 nm, 4.8 mW/cm2), even with short light-exposure times (10-60 min). In contrast, the mononuclear Pt-OH and Ir-NH2 subunits and their simple mixture are much less potent. Cellular Pt accumulation was higher for Pt-Ir compared to Pt-OH. Irradiation of Pt-Ir in cancer cells damages nuclei and releases chromosomes. Synchrotron-XRF revealed ca. 4× higher levels of intracellular platinum compared to iridium in Pt-Ir treated cells under dark conditions. Luminescent Pt-Ir distributes over the whole cell and generates ROS and 1O2 within 1 h of irradiation. Iridium localises strongly in small compartments, suggestive of complex cleavage and excretion via recycling vesicles (e.g. lysosomes). The combination of PDT and PACT motifs in one molecule, provides Pt-Ir with a novel strategy for multimodal phototherapy.

An uneven distribution of strontium in the coccolithophore Scyphosphaera apsteinii revealed by nanoscale X-ray fluorescence tomography.

Coccolithophores are biogeochemically and ecologically important phytoplankton that produce a composite calcium carbonate-based exoskeleton - the coccosphere - comprised of individual platelets, known as coccoliths. Coccoliths are stunning examples of biomineralization; their formation featuring exceptional control over both biomineral chemistry and shape. Understanding how coccoliths are formed requires information about minor element distribution and chemical environment. Here, the first high-resolution 3D synchrotron X-ray fluorescence (XRF) mapping of a coccolith is presented, showing that the lopadoliths of Scyphosphaera apsteinii display stripes of different Sr concentration. The presence of Sr stripes is unaffected by elevated Sr in the culture medium, macro-nutrient concentration, and light intensity, indicating that the observed stripiness is an expression of the fundamental coccolith formation process in this species. Current Sr fractionation models, by contrast, predict an even Sr distribution and will have to be modified to account for this stripiness. Additionally, nano-XANES analyses show that Sr resides in a Ca site in the calcite lattice in both high and low Sr stripes, confirming a central assumption of current Sr fractionation models.

Toward Understanding the Environmental Risks of Combined Microplastics/Nanomaterials Exposures: Unveiling ZnO Transformations after Adsorption onto Polystyrene Microplastics in Environmental Solutions.

Over recent decades, there has been a dramatic increase in the manufacture of engineered nanomaterials, which has inevitably led to their environmental release. Zinc oxide (ZnO) is among the more abundant nanomaterial manufactured due to its advantageous properties, used for piezoelectric, semiconducting, and antibacterial purposes. Plastic waste is ubiquitous and may break down or delaminate into smaller microplastics, leaving open the question of whether these small polymers may alter the fate of ZnO through adsorption within aquatic media (tap-water and seawater). Here, scanning electron microscopy analysis confirms the effective Zn nano/microstructures adsorption onto polystyrene surfaces after only 24-h incubation in the aquatic media. After pre-aging the nanomaterials for 7-days in different environmental media, nanoprobe X-ray absorption near-edge spectroscopy analysis reveals significant ZnO transformation toward Zn-sulfide and Zn-phosphate. The interaction between a commercial ZnO-based sunscreen with polystyrene and a cleanser consumer containing microbeads with ZnO nanomaterials is also studied, revealing the adsorption of transformed Zn-species in the microplastics surfaces, highlighting the environmental relevancy of this work. Understanding the structural and functional impacts of the microplastics/ZnO complexes, and how they evolve, will provide insights into their chemical nature, stability, transformations, and fate, which is key to predicting their bioreactivity in the environment.

Differential phase contrast for quantitative imaging and spectro-microscopy at a nanoprobe beamline.

The interaction of a focused X-ray beam with a sample in a scanning probe experiment can provide a variety of information about the interaction volume. In many scanning probe experiments X-ray fluorescence (XRF) is supplemented with measurements of the transmitted or scattered intensity using a pixelated detector. The automated extraction of different signals from an area pixelated detector is described, in particular the methodology for extracting differential phase contrast (DPC) is demonstrated and different processing methods are compared across a range of samples. The phase shift of the transmitted X-ray beam by the sample, extracted from DPC, is also compared with ptychography measurements to provide a qualitative and quantitative comparison. While ptychography produces a superior image, DPC can offer a simple, flexible method for phase contrast imaging which can provide fast results and feedback during an experiment; furthermore, for many science problems, such as registration of XRF in a lighter matrix, DPC can provide sufficient information to meet the experimental aims. As the DPC technique is a quantitative measurement, it can be expanded to spectroscopic studies and a demonstration of DPC for spectro-microscopy measurements is presented. Where ptychography can separate the absorption and phase shifts by the sample, quantitative interpretation of a DPC image or spectro-microscopy signal can only be performed directly when absorption is negligible or where the absorption contribution is known and the contributions can be fitted.

The Delta Robot-A long travel nano-positioning stage for scanning x-ray microscopy.

A new stage design concept, the Delta Robot, is presented, which is a parallel kinematic design for scanning x-ray microscopy applications. The stage employs three orthogonal voice coils, which actuate parallelogram flexures. The design has a 3 mm travel range and achieves rms position jitter, integrated from 1 Hz to 1 kHz, of 2.8 and 1.3 nm perpendicular to the beam and 5.6 nm along the beam direction with loads up to 350 g. The Delta Robot design process used a mechatronics approach of iterative modeling and simulation to develop the system and validate performance. The design considerations, design process, stability, and operational performance on the hard x-ray nanoprobe at Diamond Light Source are presented.

A cell design for correlative hard X-ray nanoprobe and electron microscopy studies of catalysts under in situ conditions.

To improve the understanding of catalysts, and ultimately the ability to design better materials, it is crucial to study them during their catalytic active states. Using in situ or operando conditions allows insights into structure-property relationships, which might not be observable by ex situ characterization. Spatially resolved X-ray fluorescence, X-ray diffraction and X-ray absorption near-edge spectroscopy are powerful tools to determine structural and electronic properties, and the spatial resolutions now achievable at hard X-ray nanoprobe beamlines make them an ideal complement to high-resolution transmission electron microscopy studies in a multi-length-scale analysis approach. The development of a system to enable the use of a commercially available gas-cell chip assembly within an X-ray nanoprobe beamline is reported here. The novel in situ capability is demonstrated by an investigation of the redox behaviour of supported Pt nanoparticles on ceria under typical lean and rich diesel-exhaust conditions; however, the system has broader application to a wide range of solid-gas reactions. In addition the setup allows complimentary in situ transmission electron microscopy and X-ray nanoprobe studies under identical conditions, with the major advantage compared with other systems that the exact same cell can be used and easily transferred between instruments. This offers the exciting possibility of studying the same particles under identical conditions (gas flow, pressure, temperature) using multiple techniques.

Nanoscale chemical heterogeneity dominates the optoelectronic response of alloyed perovskite solar cells.

Halide perovskites perform remarkably in optoelectronic devices. However, this exceptional performance is striking given that perovskites exhibit deep charge-carrier traps and spatial compositional and structural heterogeneity, all of which should be detrimental to performance. Here, we resolve this long-standing paradox by providing a global visualization of the nanoscale chemical, structural and optoelectronic landscape in halide perovskite devices, made possible through the development of a new suite of correlative, multimodal microscopy measurements combining quantitative optical spectroscopic techniques and synchrotron nanoprobe measurements. We show that compositional disorder dominates the optoelectronic response over a weaker influence of nanoscale strain variations even of large magnitude. Nanoscale compositional gradients drive carrier funnelling onto local regions associated with low electronic disorder, drawing carrier recombination away from trap clusters associated with electronic disorder and leading to high local photoluminescence quantum efficiency. These measurements reveal a global picture of the competitive nanoscale landscape, which endows enhanced defect tolerance in devices through spatial chemical disorder that outcompetes both electronic and structural disorder.

Single-Cell Chemistry of Photoactivatable Platinum Anticancer Complexes.

The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species.

Beam and sample movement compensation for robust spectro-microscopy measurements on a hard X-ray nanoprobe.

Static and in situ nanoscale spectro-microscopy is now routinely performed on the Hard X-ray Nanoprobe beamline at Diamond and the solutions implemented to provide robust energy scanning and experimental operation are described. A software-based scheme for active feedback stabilization of X-ray beam position and monochromatic beam flux across the operating energy range of the beamline is reported, consisting of two linked feedback loops using extremum seeking and position control. Multimodal registration methods have been implemented for active compensation of drift during an experiment to compensate for sample movement during in situ experiments or from beam-induced effects.

The Hard X-ray Nanoprobe beamline at Diamond Light Source.

The Hard X-ray Nanoprobe beamline, I14, at Diamond Light Source is a new facility for nanoscale microscopy. The beamline was designed with an emphasis on multi-modal analysis, providing elemental mapping, speciation mapping by XANES, structural phase mapping using nano-XRD and imaging through differential phase contrast and ptychography. The 185 m-long beamline operates over a 5 keV to 23 keV energy range providing a ≤50 nm beam size for routine user experiments and a flexible scanning system allowing fast acquisition. The beamline achieves robust and stable operation by imaging the source in the vertical direction and implementing horizontally deflecting primary optics and an overfilled secondary source in the horizontal direction. This paper describes the design considerations, optical layout, aspects of the hardware engineering and scanning system in operation as well as some examples illustrating the beamline performance.

Tracking Reactions of Asymmetric Organo-Osmium Transfer Hydrogenation Catalysts in Cancer Cells.

Most metallodrugs are prodrugs that can undergo ligand exchange and redox reactions in biological media. Here we have investigated the cellular stability of the anticancer complex [OsII [(η6 -p-cymene)(RR/SS-MePh-DPEN)] [1] (MePh-DPEN=tosyl-diphenylethylenediamine) which catalyses the enantioselective reduction of pyruvate to lactate in cells. The introduction of a bromide tag at an unreactive site on a phenyl substituent of Ph-DPEN allowed us to probe the fate of this ligand and Os in human cancer cells by a combination of X-ray fluorescence (XRF) elemental mapping and inductively coupled plasma-mass spectrometry (ICP-MS). The BrPh-DPEN ligand is readily displaced by reaction with endogenous thiols and translocated to the nucleus, whereas the Os fragment is exported from the cells. These data explain why the efficiency of catalysis is low, and suggests that it could be optimised by developing thiol resistant analogues. Moreover, this work also provides a new way for the delivery of ligands which are inactive when administered on their own.

X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser.

The organoiridium complex Ir[(C,N)2(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (1O2) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of 1O2 generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.

A passive hutch-cooling system for achieving high thermal-stability operation at the Nanoprobe beamline, Diamond Light Source.

The development of low-emittance storage rings and the rapid developments in nano-optics and imaging techniques are leading to decreasing X-ray spot sizes and increasing requirements on the environmental and mechanical stability of beamline components. In particular, temperature stability in the experimental hutches is critical to minimize uncontrolled displacements caused by thermal expansion and ensure consistent performance. Here, the design and thermal performance of the experimental hutches of the Nanoprobe beamline at Diamond Light Source are described, where a standard deviation of the room temperature down to 0.017°C over extended periods is demonstrated. The rooms are kept at constant temperature using water-cooled radiant panels which line the ceiling and walls. Radiant panels are relatively common in high-end electron microscopy rooms, but this is the first demonstration of their use for fine temperature control in an X-ray hutch and may provide a useful basis for future upgrades at upcoming low-emittance sources.

Spatially Resolved Dissolution and Speciation Changes of ZnO Nanorods during Short-Term in Situ Incubation in a Simulated Wastewater Environment.

Zinc oxide engineered nanomaterials (ZnO ENMs) are used in a variety of applications worldwide due to their optoelectronic and antibacterial properties with potential contaminant risk to the environment following their disposal. One of the main potential pathways for ZnO nanomaterials to reach the environment is via urban wastewater treatment plants. So far there is no technique that can provide spatiotemporal nanoscale information about the rates and mechanisms by which the individual nanoparticles transform. Fundamental knowledge of how the surface chemistry of individual particles change, and the heterogeneity of transformations within the system, will reveal the critical physicochemical properties determining environmental damage and deactivation. We applied a methodology based on spatially resolved in situ X-ray fluorescence microscopy (XFM), allowing observation of real-time dissolution and morphological and chemical evolution of synthetic template-grown ZnO nanorods (∼725 nm length, ∼140 nm diameter). Core-shell ZnO-ZnS nanostructures were formed rapidly within 1 h, and significant amounts of ZnS species were generated, with a corresponding depletion of ZnO after 3 h. Diffuse nanoparticles of ZnS, Zn3(PO4)2, and Zn adsorbed to Fe-oxyhydroxides were also imaged in some nonsterically impeded regions after 3 h. The formation of diffuse nanoparticles was affected by ongoing ZnO dissolution (quantified by inductively coupled plasma mass spectrometry) and the humic acid content in the simulated sludge. Complementary ex situ X-ray absorption spectroscopy and scanning electron microscopy confirmed a significant decrease in the ZnO contribution over time. Application of time-resolved XFM enables predictions about the rates at which ZnO nanomaterials transform during their first stages of the wastewater treatment process.

The Chemical Form of Metal Species Released from Corroded Taper Junctions of Hip Implants: Synchrotron Analysis of Patient Tissue.

The mechanisms of metal release from the articulation at the head cup bearing and the tapered junctions of orthopaedic hip implants are known to differ and the debris generated varies in size, shape and volume. Significantly less metal is lost from the taper junction between Cobalt-Chromium-Molybdenum (CoCrMo) and Titanium (Ti) components (fretting-corrosion dominant mechanism), when compared to the CoCrMo bearing surfaces (wear-corrosion dominant mechanism). Corrosion particles from the taper junction can lead to Adverse Reactions to Metal Debris (ARMD) similar to those seen with CoCrMo bearings. We used synchrotron methods to understand the modes underlying clinically significant tissue reactions to Co, Cr and Ti by analysing viable peri-prosthetic tissue. Cr was present as Cr2O3 in the corroded group in addition to CrPO4 found in the metal-on-metal (MoM) group. Interestingly, Ti was present as TiO2 in an amorphous rather than rutile or anatase physical form. The metal species were co-localized in the same micron-scale particles as result of corrosion processes and in one cell type, the phagocytes. This work gives new insights into the degradation products from metal devices as well as guidance for toxicological studies in humans.

Automatic processing of multimodal tomography datasets.

With the development of fourth-generation high-brightness synchrotrons on the horizon, the already large volume of data that will be collected on imaging and mapping beamlines is set to increase by orders of magnitude. As such, an easy and accessible way of dealing with such large datasets as quickly as possible is required in order to be able to address the core scientific problems during the experimental data collection. Savu is an accessible and flexible big data processing framework that is able to deal with both the variety and the volume of data of multimodal and multidimensional scientific datasets output such as those from chemical tomography experiments on the I18 microfocus scanning beamline at Diamond Light Source.

Detection of metallic cobalt and chromium liver deposition following failed hip replacement using T2* and R2 magnetic resonance.

BACKGROUND: Failed hip prostheses can cause elevated circulating cobalt and chromium levels, with rare reports of fatal systemic organ deposition, including cobalt cardiomyopathy. Although blood cobalt and chromium levels are easily measured, organ deposition is difficult to detect without invasive biopsy. The T2* magnetic resonance (MR) method is used to quantify tissue iron deposition, and plays an important role in the management of iron-loading conditions. Cobalt and chromium, like iron, also affect magnetism and are proposed MR contrast agents. CASE PRESENTATION: We describe a case of a 44-year-old male with a failed hip implant and very elevated blood cobalt and chromium levels. Despite normal cardiac MR findings, liver T2* and R2 values were abnormal, triggering tissue biopsy. Liver tissue analysis, including X-ray fluorescence, demonstrated heavy elemental cobalt and chromium deposition in macrophages, and no detectable iron. CONCLUSIONS: Our case demonstrates T2* and R2 quantification of liver metal deposition in a patient with a failed hip implant. Further work is needed to investigate the role of T2* and R2 MR in the detection of metal deposition from metal on metal hip prostheses.

Understanding the tissue effects of tribo-corrosion: uptake, distribution, and speciation of cobalt and chromium in human bone cells.

Cobalt and chromium species are released in the local tissues as a result of tribo-corrosion, and affect bone cell survival and function. However we have little understanding of the mechanisms of cellular entry, intracellular distribution, and speciation of the metals that result in impaired bone health. Here we used synchrotron based X-ray fluorescence (XRF), X-ray absorption spectroscopy (XAS), and fluorescent-probing approaches of candidate receptors P2X7R and divalent metal transporter-1 (DMT-1), to better understand the entry, intra-cellular distribution and speciation of cobalt (Co) and chromium (Cr) in human osteoblasts and primary human osteoclasts. We found that both Co and Cr were most highly localized at nuclear and perinuclear sites in osteoblasts, suggesting uptake through cell membrane transporters, and supported by a finding that P2X7 receptor blockade reduced cellular entry of Co. In contrast, metal species were present at discrete sites corresponding to the basolateral membrane in osteoclasts, suggesting cell entry by endocytosis and trafficking through a functional secretory domain. An intracellular reduction of Cr6+ to Cr3+ was the only redox change observed in cells treated with Co2+, Cr3+, and Cr6+. Our data suggest that the cellular uptake and processing of Co and Cr differs between osteoblasts and osteoclasts.

Combining µXANES and µXRD mapping to analyse the heterogeneity in calcium carbonate granules excreted by the earthworm Lumbricus terrestris.

The use of fluorescence full spectral micro-X-ray absorption near-edge structure (µXANES) mapping is becoming more widespread in the hard energy regime. This experimental method using the Ca K-edge combined with micro-X-ray diffraction (µXRD) mapping of the same sample has been enabled on beamline I18 at Diamond Light Source. This combined approach has been used to probe both long- and short-range order in calcium carbonate granules produced by the earthworm Lumbricus terrestris. In granules produced by earthworms cultured in a control artificial soil, calcite and vaterite are observed in the granules. However, granules produced by earthworms cultivated in the same artificial soil amended with 500 p.p.m. Mg also contain an aragonite. The two techniques, µXRD and µXANES, probe different sample volumes but there is good agreement in the phase maps produced.

How does iron interact with sporopollenin exine capsules? An X-ray absorption study including microfocus XANES and XRF imaging.

Sporopollenin exine capsules (SECs) derived from plant spores and pollen grains have been proposed as adsorption, remediation and drug delivery agents. Despite many studies there is scant structural data available. This X-ray absorption investigation represents the first direct structural data on the interaction of metals with SECs and allows elucidation of their structure-property relationships. Fe K-edge XANES and EXAFS data have shown that the iron local environment in SECs (derived from Lycopodium clavatum) reacted with aqueous ferric chloride solutions is similar to that of ferrihydrite (FeOOH) and by implication ferritin. Fe Kα XRF micro-focus experiments show that there is a poor correlation between the iron distribution and the underlying SEC structure indicating that the SEC is coated in the FeOOH material. In contrast, the Fe Kα XRF micro-focus experiments on SECs reacted with aqueous ferrous chloride solutions show that there is a very high correlation between the iron distribution and the SEC structure, indicating a much more specific form of interaction of the iron with the SEC surface functional groups. Fe K-edge XANES and EXAFS data show that the FeII can be easily oxidised to give a structure similar to, but not identical to that in the FeIII case, and that even if anaerobic conditions are used there is still partial oxidation to FeIII.