Sofia Diaz-Moreno

We report the technical design, implementation and operation of a newly developed X-ray emission spectrometer on the I20 beamline at Diamond Light Source. The spectrometer consists of 14 crystal analysers arranged in an up–down configuration, allowing for operation in one- or two-colour acquisition modes. Since beginning operations in 2023, the spectrometer has substantially enhanced the capability of the beamline to perform X-ray emission spectroscopy (XES) and has demonstrated high reliability with minimal operational issues during user experiments. The latter achievement is particularly significant given the complexity of the instrument, and the difficulty of maintaining the Rowland condition when scanning the energy. We show that the spectrometer can effectively measure spectra in two-colour mode and is capable of detecting weak valence-to-core emission features with a significantly improved signal-to-background ratio. We also present data taken using a newly developed quick-scanning XES acquisition mode, which enables data collection in seconds rather than minutes. This mode opens up possibilities for time-resolved studies and investigating radiation-sensitive materials.

Metals play an essential role in cellular homeostasis and are key components of several formulations currently used in the clinic. Synchrotron‐based X‐ray microscopy at submicron resolution is a powerful approach to map intracellular elemental distributions and to monitor how these patterns change upon genetic or pharmacological perturbations. However, existing sample‐preparation protocols often rely on costly and highly specialized equipment for vitrification and dehydration, limiting their widespread adoption. Here, we present an adapted plunge‐freezing and freeze‐drying workflow that enables the preparation of mammalian cell samples for X‐ray fluorescence (XRF) and X‐ray absorption spectroscopy (XAS) studies with submicron resolution in a cost‐effective and versatile manner. Furthermore, we define acquisition parameters optimized for the reliable detection of low‐abundance metals, such as endogenous iron. We anticipate that this accessible protocol will facilitate the broader implementation of synchrotron‐based inner‐shell spectromicroscopy in cell biology.

Nucleophosmin (NPM1) is a nucleolar protein commonly mutated in ~30% of newly diagnosed acute myeloid leukemia (AML) cases. These mutations occur in the terminal exon of the NPM1 gene, affecting the C-terminal DNA-binding domain of the protein and causing its delocalization to the cytoplasm—a hallmark of NPM1-mutated AML. NPM1 shuttling to the nucleoplasm is tightly regulated by posttranslational modifications, such as phosphorylation of Ser254, Ser260, and Tyr271 of the DNA-binding domain. However, the structural mechanisms underlying this process remain unclear. In this work, we show that Ser-to-Asp (S254D–S260D) and Tyr-to-pCMF (para-carboxymethyl phenylalanine) (Y271pCMF) phosphomimetic mutations induce significant structural and dynamical rearrangements, as well as drastic modifications in electrostatic surface potential. These changes compromise recognition of a G-quadruplex sequence from the c-MYC promoter by reducing DNA-binding affinity, reshape histone capturing dynamics, and fade charge segregation in the histone-binding domain. Combination of such substitutions in a triple phosphomimetic variant (S254D–S260D–Y271pCMF) further destabilizes the domain’s structure and triggers protein aggregation. Altogether, these findings suggest that phosphorylation of Ser254, Ser260, and Tyr271 of the C-end DNA-binding domain weakens both DNA affinity and charge block-driven liquid–liquid phase separation, offering a molecular explanation for the delocalization of NPM1 outside of the nucleolus.

The discovery of the novel n = 2 satellite transition in the Kβ emission spectrum of manganese and its evolution with incident photon energy are presented. Using the XR-HERFD (extended-range high-energy-resolution fluorescence detection) technique, we conclusively demonstrate the existence of this phenomenon with a statistical significance corresponding to 652 σ_se across the measured spectrum, far above the discovery threshold of 3–6 σ_se. We apply principal component analysis (PCA) to the XR-HERFD data to extract advanced structural insights. The evolution of this novel spectral feature and physical process are quantified by incorporating regression, revealing the increase in intensity over a wide range of incident photon energies. We validate these findings through independent test data. These results directly challenge the conventional treatment of the many-body reduction factor S ₀ ² as a constant independent of incident photon energy in the standard XAFS (X-ray absorption fine structure) equation. Thereby, these results present compelling evidence that S ₀ ² should be modelled as a varying function of incident photon energy, marking the first observation of this behaviour in Kβ spectra. This facilitates a greater quantitative understanding of HERFD spectra and a comprehensive representation of many-body effects in condensed matter systems.

This study of manganese (Mn, Z = 25) introduces a novel combination of extended-range high energy resolution fluorescence detection (XR-HERFD), multiple-crystal spectrometers and advanced binary data splicing techniques to address challenges in X-ray emission spectroscopy. XR-HERFD enhances spectral precision by utilizing high-resolution crystal analysers and optimized detector configurations. The systematic application of these methods using multiple Bragg crystal analysers at Diamond Light Source has led to substantial improvements in data quality. Simultaneously, advanced binary data splicing integrates multiple datasets to correct distortions and improve resolution, resulting in sharper spectral features. Our results show a significant increase in peak counts and a notable reduction in full width at half-maximum (FWHM), with peak amplitudes increasing by 83% and resolution improving by 46%. These developments provide greater detail for X-ray absorption or emission spectra, offering valuable insights into complex materials, and permitting advances and breakthroughs in atomic relativistic quantum mechanics, chemical sensitivity of atomic transitions and modelling of solid-state effects.

The technique of extended-range high-energy-resolution fluorescence detection (XR-HERFD), developed from X-ray absorption spectroscopy, X-ray emission spectroscopy and resonant inelastic X-ray scattering (RIXS), has been used to successfully observe a new X-ray fluorescent satellite in manganese. The experimental methodology, spectral processing and analysis, and how statistical information and structure can be defined, extracted and used from HERFD spectra are detailed. Novel approaches to measure and improve accurate data uncertainty in XR-HERFD, HERFD and RIXS data sets are also presented. This includes definitions of intrinsic resolution and improvements to the resolution of the output and data by a factor of two relative to raw data or standard processing. Novel systematics common in HERFD and RIXS experiments are detailed, including background subtraction and elastic Bragg harmonics, with approaches to dealing with them.

Understanding irradiation damage of REBCO is increasingly of interest for compact tokamak fusion reactor designs, as these materials are critical for the proposed magnetic plasma confinement systems. Here commercially sourced samples of REBCO coated conductor are irradiated with 300 keV He⁺ ions to a damage level of 169 × 10⁻³ displacements-per-atom, to the point where superconductivity is no longer detectable, meaning these samples correspond to a non-functional end-of-life component in a fusion reactor context. Subsequent analysis of the crystal structure through a combination of x-ray diffraction and x-ray absorption spectroscopy measurements reveals a complex variation away from the as-grown structure. The local structure probed by the spectroscopy measurements is further observed to change as a function of the relative polarisation of the incident x-ray beam, indicating that within this damage regime the structural anisotropy of the REBCO unit cell plays a determining role in where defects accumulate within the material. Here the local structure measurements probing the a-b plane of the system vary significantly less than those probing the c-axis direction following irradiation, mirroring the observed trend in the x-ray diffraction data that the a:b ratio is preserved upon irradiation whilst the absolute values increase, whereas the c-axis parameter expands. These observations highlight the role of oxygen defect formation in driving the degradation of superconductivity within irradiated REBCO. These changes are observed to preferentially accumulate along the c-axis of the material, indicating a possible mechanistic signature of the degradation of the superconducting properties within these systems that are evident using a local structure probe such as extended x-ray absorption fine structure.

A free-standing and compact reaction cell for combined in situ/operando x-ray spectroscopy, scattering, and imaging measurements at high pressures and high temperatures is described. The cell permits measurements under realistic operating conditions (up to 50 bar and 1000 °C), under static and flow conditions (up to 100 ml/min), over a wide range of hard x-ray energies, variable detection modes (transmission, fluorescence, and scattering), and at all angles of rotation. An operando XAS, x-ray fluorescence, x-ray computed tomography, and x-ray diffraction computed tomography case study on the reduction of a heterogeneous catalyst is presented to illustrate the performance of the reaction cell.