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Understanding the osteochondral junction, where non-mineralised cartilage and mineralised bone converge, is crucial for joint health. Current sample preparation techniques are insufficient for detailed spatial hyperspectral imaging analysis. Using the enhanced Kawamoto method, we used the super cryo embedding medium’s temperature-dependent properties to transfer high-quality tissue samples onto slides for spatial imaging analysis. We transferred osteochondral samples using a tape-free system and successfully tested them in hematoxylin and eosin (HE), Safranin-O, nanomechanical assessments and nano-Fourier transform infrared (FTIR) mapping. This protocol elucidates the structural and elemental gradients, mechanical characteristics and distinctive biochemical layering, making it a useful tool for analysing biochemical properties’ co-distribution in healthy and diseased situations. [ABSTRACT FROM AUTHOR]

X-ray ptychography is a coherent diffraction imaging technique that allows for the quantitative retrieval of both the amplitude and phase information of a sample in diffraction-limited resolution. However, traditional reconstruction algorithms require a large number of iterations to obtain phase and amplitude images exactly, and the expensive computation precludes real-time imaging. To solve the inverse problem of ptychography data, PtychoNN uses deep convolutional neural networks for real-time imaging. However, its model is relatively simple, and its accuracy is limited by the size of the training dataset, resulting in lower robustness. To address this problem, a series of W-Net neural network models have been proposed which can robustly reconstruct the object phase information from the raw data. Numerical experiments demonstrate that our neural network exhibits better robustness, superior reconstruction capabilities and shorter training time with high-precision ptychography imaging. [ABSTRACT FROM AUTHOR]

X-ray ptychography is a coherent diffraction imaging technique that allows for the quantitative retrieval of both the amplitude and phase information of a sample in diffraction-limited resolution. However, traditional reconstruction algorithms require a large number of iterations to obtain phase and amplitude images exactly, and the expensive computation precludes real-time imaging. To solve the inverse problem of ptychography data, PtychoNN uses deep convolutional neural networks for real-time imaging. However, its model is relatively simple, and its accuracy is limited by the size of the training dataset, resulting in lower robustness. To address this problem, a series of W-Net neural network models have been proposed which can robustly reconstruct the object phase information from the raw data. Numerical experiments demonstrate that our neural network exhibits better robustness, superior reconstruction capabilities and shorter training time with high-precision ptychography imaging.

In the realm of X-ray ptychography experiments, a considerable amount of ptychography scans are typically performed within a field of view encompassing the target sample. While it is crucial to obtain overlapping scans in small increments over the region of interest for achieving high-resolution sample reconstruction, a significant number of these scans often redundantly measure the empty background within the wide field of view. To address this inefficiency, an innovative algorithm is proposed that introduces automatic guidance for data acquisition. The algorithm first directs the scan point to actively search for the object of interest within the field of view. Subsequently, it intelligently scans along the perimeter of the sample, strategically acquiring measurements exclusively within the boundary of the region of interest. By employing this approach, a reduction in the number of measurements required to obtain high-resolution reconstruction images is demonstrated, as compared with conventional raster scanning methods. Furthermore, the automatic guidance provided by the algorithm offers the added advantage of saving valuable time during the reconstruction process. Through practical implementation on real experiments, these findings showcase the efficacy of the proposed algorithm in enhancing the efficiency and accuracy of X-ray ptychography experiments. This novel approach holds immense potential for advancing sample analysis and imaging techniques in various scientific disciplines.

Lithium iron phosphate (LixFePO4), a cathode material used in rechargeable Li-ion batteries, phase separates upon de/lithiation under equilibrium. The interfacial structure and chemistry within these cathode materials affects Li-ion transport, and therefore battery performance. Correlative imaging of LixFePO4 was performed using four-dimensional scanning transmission electron microscopy (4D-STEM), scanning transmission X-ray microscopy (STXM), and X-ray ptychography in order to analyze the local structure and chemistry of the same particle set. Over 50,000 diffraction patterns from 10 particles provided measurements of both structure and chemistry at a nanoscale spatial resolution (16.6–49.5 nm) over wide (several micron) fields-of-view with statistical robustness. LixFePO4 particles at varying stages of delithiation were measured to examine the evolution of structure and chemistry as a function of delithiation. In lithiated and delithiated particles, local variations were observed in the degree of lithiation even while local lattice structures remained comparatively constant, and calculation of linear coefficients of chemical expansion suggest pinning of the lattice structures in these populations. Partially delithiated particles displayed broadly core–shell-like structures, however, with highly variable behavior both locally and per individual particle that exhibited distinctive intermediate regions at the interface between phases, and pockets within the lithiated core that correspond to FePO4 in structure and chemistry. The results provide insight into the LixFePO4 system, subtleties in the scope and applicability of Vegard's law (linear lattice parameter-composition behavior) under local versus global measurements, and demonstrate a powerful new combination of experimental and analytical modalities for bridging the crucial gap between local and statistical characterization.

This study reports the characterisation of human dental enamel caries using synchrotron nanoscale correlative ptychography and spectroscopic mapping in combination with scanning electron microscopy. A lamella ̴2.4 µm thick was extracted from a carious enamel region of a tooth using focused ion beam-scanning electron microscopy and transferred to two synchrotron beamlines to perform hard X-ray nano-fluorescence spectroscopy simultaneously with differential phase contrast mapping at a beam size of 55 × 45 nm. Soft X-ray ptychography data was then reconstructed with a pixel size of 8 nm. The two dimensional variation in chemistry and structure of carious enamel was revealed at the nanoscale, namely, the organisation of hydroxyapatite nano-crystals within enamel rods was imaged together with the inter-rod region. Correlative use of electron and X-ray scanning microscopies for the same sample allowed visualisation of the connection between structure and composition as presented in a compound image where colour indicates the relative calcium concentration in the sample, as indicated by the calcium Kα fluorescence intensity and grey scale shows the nanostructure. This highlights the importance of advanced correlative imaging to investigate the complex structure-composition relationships in nanomaterials of natural or artificial origin.

The spectroscopic ptychography method, a technique combining X-ray ptychography imaging and X-ray absorption spectroscopy, is one of the most promising and powerful tools for studying the chemical states and morphological structures of bulk materials at high resolutions. However, this technique still requires long measurement periods because of insufficient coherent X-ray intensity. Although the improvements in hardware represent a critical solution, breakthroughs in software for experiments and analyses are also required. This paper proposes a novel method for restoring the spectrum structures from spectroscopic ptychography measurements with reduced energy points, by utilizing the Kramers--Kronig relationship. First, a numerical simulation is performed of the spectrum restoration for the extended X-ray absorption fine structure (EXAFS) oscillation from the thinned theoretical absorption and phase spectra. Then, this algorithm is extended by binning the noise removal to handle actual experimental spectral data. Spectrum restoration for the experimental EXAFS data obtained from spectroscopic ptychography measurements is also successfully demonstrated. The proposed restoration will help shorten the time required for spectroscopic ptychography single measurements and increase the throughput of the entire experiment under limited time resources. [ABSTRACT FROM AUTHOR]

The synthesis of hierarchically porous materials usually requires complex experimental procedures, often based around extensive trial and error approaches. One common synthesis strategy is the sol–gel method, although the relation between synthesis parameters, material structure and function has not been widely explored. Here, in situ 2D hard X‐ray ptychography (XRP) and 3D ptychographic X‐ray computed tomography (PXCT) are applied to monitor the development of hierarchical porosity in Ni/Al2O3 and Al2O3 catalysts with connected meso‐ and macropore networks. In situ XRP allows to follow textural changes of a dried gel Ni/Al2O3 sample as a function of temperature during calcination, activation and CO2 methanation reaction. Complementary PXCT studies on dried gel particles of Ni/Al2O3 and Al2O3 provide quantitative information on pore structure, size distribution, and shape with 3D spatial resolution approaching 50 nm, while identical particles are imaged ex situ before and after calcination. The X‐ray imaging results are correlated with N2‐sorption, Hg porosimetry and He pycnometry pore characterization. Hard X‐ray nanotomography is highlighted to derive fine structural details including tortuosity, branching nodes, and closed pores, which are relevant in understanding transport phenomena during chemical reactions. XRP and PXCT are enabling technologies to understand complex synthesis pathways of porous materials. [ABSTRACT FROM AUTHOR]

[Display omitted] Lithium iron phosphate (Li x FePO 4), a cathode material used in rechargeable Li-ion batteries, phase separates upon de/lithiation under equilibrium. The interfacial structure and chemistry within these cathode materials affects Li-ion transport, and therefore battery performance. Correlative imaging of Li x FePO 4 was performed using four-dimensional scanning transmission electron microscopy (4D-STEM), scanning transmission X-ray microscopy (STXM), and X-ray ptychography in order to analyze the local structure and chemistry of the same particle set. Over 50,000 diffraction patterns from 10 particles provided measurements of both structure and chemistry at a nanoscale spatial resolution (16.6–49.5 nm) over wide (several micron) fields-of-view with statistical robustness. Li x FePO 4 particles at varying stages of delithiation were measured to examine the evolution of structure and chemistry as a function of delithiation. In lithiated and delithiated particles, local variations were observed in the degree of lithiation even while local lattice structures remained comparatively constant, and calculation of linear coefficients of chemical expansion suggest pinning of the lattice structures in these populations. Partially delithiated particles displayed broadly core–shell-like structures, however, with highly variable behavior both locally and per individual particle that exhibited distinctive intermediate regions at the interface between phases, and pockets within the lithiated core that correspond to FePO 4 in structure and chemistry. The results provide insight into the Li x FePO 4 system, subtleties in the scope and applicability of Vegard's law (linear lattice parameter-composition behavior) under local versus global measurements, and demonstrate a powerful new combination of experimental and analytical modalities for bridging the crucial gap between local and statistical characterization. [ABSTRACT FROM AUTHOR]

X‐ray ptychography and X‐ray fluorescence are complementary nanoscale imaging techniques, providing structural and elemental information, respectively. Both methods acquire data by scanning a localized beam across the sample. X‐ray ptychography processes the transmission signal of a coherent illumination interacting with the sample, to produce images with a resolution finer than the illumination spot and step size. By enlarging both the spot and the step size, the technique can cover extended regions efficiently. X‐ray fluorescence records the emitted spectra as the sample is scanned through the localized beam and its spatial resolution is limited by the spot and step size. The requisites for fast ptychography and high‐resolution fluorescence appear incompatible. Here, a novel scheme that mitigates the difference in requirements is proposed. The method makes use of two probes of different sizes at the sample, generated by using two different energies for the probes and chromatic focusing optics. The different probe sizes allow to reduce the number of acquisition steps for the joint fluorescence–ptychography scan compared with a standard single beam scan, while imaging the same field of view. The new method is demonstrated experimentally using two undulator harmonics, a Fresnel zone plate and an energy discriminating photon counting detector. [ABSTRACT FROM AUTHOR]

Understanding catalyst deactivation by coking is crucial for knowledge‐based catalyst and process design in reactions with carbonaceous species. Post‐mortem analysis of catalyst coking is often performed by bulk characterization methods. Here, hard X‐ray ptychographic computed tomography (PXCT) was used to study Ni/Al2O3 catalysts for CO2 methanation and CH4 dry reforming after artificial coking treatment. PXCT generated quantitative 3D maps of local electron density at ca. 80 nm resolution, allowing to visualize and evaluate the severity of coking in entire catalyst particles of ca. 40 μm diameter. Coking was primarily revealed in the nanoporous solid, which was not detectable in resolved macropores. Coke formation was independently confirmed by operando Raman spectroscopy. PXCT is highlighted as an emerging characterization tool for nanoscale identification, co‐localization, and potentially quantification of deactivation phenomena in 3D space within entire catalyst particles. [ABSTRACT FROM AUTHOR]

Increases in X‐ray brightness from synchrotron light sources lead to a requirement for higher frame rates from hybrid pixel array detectors (HPADs), while also favoring charge integration over photon counting. However, transfer of the full uncompressed data will begin to constrain detector design, as well as limit the achievable continuous frame rate. Here a data compression scheme that is easy to implement in a HPAD's application‐specific integrated circuit (ASIC) is described, and how different degrees of compression affect image quality in ptychography, a commonly employed coherent imaging method, is examined. Using adaptive encoding quantization, it is shown in simulations that one can digitize signals up to 16383 photons per pixel (corresponding to 14 bits of information) using only 8 or 9 bits for data transfer, with negligible effect on the reconstructed image. [ABSTRACT FROM AUTHOR]

This work describes the application of X‐ray ptychography for the inspection of complex assemblies of highly anisotropic nanocrystals embedded in a thick polymer matrix. More specifically, this case deals with CdSe/CdS octapods, with pod length L = 39 ± 2 nm and pod diameter D = 12 ± 2 nm, dispersed in free‐standing thick films (24 ± 4 µm) of polymethyl methacrylate and polystyrene, with different molecular weights. Ptychography is the only imaging method available to date that can be used to study architectures made by these types of nanocrystals in thick polymeric films, as any other alternative direct method, such as scanning/transmission electron microscopy, can be definitively ruled out as a result of the large thickness of the free‐standing films. The electron density maps of the investigated samples are reconstructed by combining iterative difference map algorithms and a maximum likelihood optimization algorithm. In addition, post image processing techniques are applied to both reduce noise and provide a better visualization of the material morphological details. Through this process, at a final resolution of 27 nm, the reconstructed maps allow us to visualize the intricate network of octapods inside the polymeric matrices. [ABSTRACT FROM AUTHOR]

This work demonstrates a combination technique of X‐ray ptychography and the extended X‐ray absorption fine structure (ptychography‐EXAFS) method, which can determine the interatomic distances of bulk materials at the nanoscale. In the high‐resolution ptychography‐EXAFS method, it is necessary to use high‐intense coherent X‐rays with a uniform wavefront in a wide energy range, hence a ptychographic measurement system installed with advanced Kirkpatrick–Baez mirror focusing optics is developed and its performance is evaluated. Ptychographic diffraction patterns of micrometre‐size MnO particles are collected by using this system at 139 energies between 6.504 keV and 7.114 keV including the Mn K absorption edge, and then the EXAFS of MnO is derived from the reconstructed images. By analyzing the EXAFS spectra obtained from a 48 nm × 48 nm region, the nanoscale bond lengths of the first and second coordination shells of MnO are determined. The present approach has great potential to elucidate the unclarified relationship among the morphology, electronic state and atomic arrangement of inhomogeneous bulk materials with high spatial resolution. [ABSTRACT FROM AUTHOR]

Biological sample-preparation procedures have been developed for imaging human chromosomes under cryogenic conditions. A new experimental setup, developed for imaging frozen samples using beamline I13 at Diamond Light Source, is described. This manuscript describes the equipment and experimental procedures as well as the authors' first ptychographic reconstructions using X-rays.

X‐ray ptychography is a coherent diffraction imaging technique with a high resolving power and excellent quantitative capabilities. Although very popular in synchrotron facilities nowadays, its implementation with X‐ray energies above 15 keV is very rare due to the challenges imposed by the high energies. Here, the implementation of high‐energy X‐ray ptychography at 17 and 33.6 keV is demonstrated and solutions to overcome the important challenges are provided. Among the particular aspects addressed are the use of an efficient high‐energy detector, a long synchrotron beamline for the high degree of spatial coherence, a beam with 1% monochromaticity providing high flux, and efficient multilayer coated Kirkpatrick–Baez X‐ray optics to shape the beam. The constraints imposed by the large energy bandwidth are carefully analyzed, as well as the requirements to sample correctly the high‐energy diffraction patterns with small speckle size. In this context, optimized scanning trajectories allow the total acquisition time to be reduced by up to 35%. The paper explores these innovative solutions at the ID16A nano‐imaging beamline by ptychographic imaging of a 200 nm‐thick gold lithography sample. [ABSTRACT FROM AUTHOR]

Multi‐slice X‐ray ptychography offers an approach to achieve images with a nanometre‐scale resolution from samples with thicknesses larger than the depth of field of the imaging system by modeling a thick sample as a set of thin slices and accounting for the wavefront propagation effects within the specimen. Here, we present an experimental demonstration that resolves two layers of nanostructures separated by 500 nm along the axial direction, with sub‐10 nm and sub‐20 nm resolutions on two layers, respectively. Fluorescence maps are simultaneously measured in the multi‐modality imaging scheme to assist in decoupling the mixture of low‐spatial‐frequency features across different slices. The enhanced axial sectioning capability using correlative signals obtained from multi‐modality measurements demonstrates the great potential of the multi‐slice ptychography method for investigating specimens with extended dimensions in 3D with high resolution. Combining multi‐slice ptychography with multi‐modality scanning probe microscopy reconstructs two planes of nanostructures separated by 500 nm with sub‐20 nm lateral resolution, assisted by simultaneously measured fluorescence maps for decoupling low‐spatial‐frequency features. [ABSTRACT FROM AUTHOR]

Utilisation of partially coherent X-rays in Coherent X-ray Diffraction Imaging (CDI) and X-ray ptychography have brought intense research and developments for the past few years. Generally, data reconstructions convergence can be achieved by considering to incorporate partially coherent X-rays in ptychographic iterative reconstructions algorithms. Recently, exploration of both X-ray probe and sample modes have been studied and it was suggested that sufficient constraints are required to achieve accurate data reconstructions due to the mathematical inseparability of multiple sample modes that are superimposed into a single set of ptychographic intensity patterns. Here we review the multi-modal ptychography that can be used as a new emerging technique to investigate dynamical sample systems with partially coherent X-rays by recent experimental and simulated demonstrations. [ABSTRACT FROM AUTHOR]

We developed a soft X-ray microscopic imaging system, CARROT, that combines ptychography and fluorescence X-ray microscopy using a total-reflection Wolter mirror illumination system. The system offers advantages such as achromaticity and long working distances, enabling seamless application to a wide wavelength range. We demonstrated high-resolution imaging of cellular structures and sub-micrometer-scale changes induced by drug administration in mammalian cells. We further showcased the capability to seamlessly switch from ptychography measurements to X-ray fluorescence measurements at different wavelengths to map elemental distributions within cells. Our findings suggest that this system can be used for investigating the correlation between intracellular structures and elemental distribution, as well as for multimodal measurements of visible light fluorescence, Raman scattering, and soft X-rays to provide valuable insights into understanding intracellular dynamics. • New imaging system combines soft X-ray ptychography & fluorescence microscopy. • High-resolution images of drug-treated mammalian cells reveal sub-micrometer changes. • Investigation of the correlation between intracellular structures and elemental distribution. [ABSTRACT FROM AUTHOR]

A method for nondestructively visualizing multisection nanostructures of integrated circuits by X-ray ptychography with a multislice approach is proposed. In this study, tilt-series ptychographic diffraction data sets of a twolayered circuit with a ~1.4 μm gap at nine incident angles are collected in a wide Q range and then artifact-reduced phase images of each layer are successfully reconstructed at ~10 nm resolution. The present method has great potential for the three-dimensional observation of flat specimens with thickness on the order of 100 μm, such as three-dimensional stacked integrated circuits based on through-silicon vias, without laborious sample preparation. [ABSTRACT FROM AUTHOR]

A combination of fast scanning chip calorimetry and X-ray ptychography is explored to study the effects of thermal annealing on the active layer of bulk heterojunction organic photovoltaics. The well-known P3HT/PC 61 BM 1:1 system is investigated as a test case. By using a custom chip calorimetry setup, it is possible to give a thermal treatment at 127 °C (400 K) to P3HT/PC 61 BM 1:1 thin layers, using a heating and cooling rate of 30000 K s −1 , after which the resulting morphology is investigated with X-ray ptychography. Applying only heating and cooling, without isothermal annealing, yields a featureless morphology. This corresponds well with thermal data which indicate a mixed amorphous phase only. For increasing isothermal annealing times, a well-defined morphology appears with increasing domain size, corresponding to the formation of an endothermal melting trajectory. This melting trajectory is expected to consist of both eutectic melting and melting of coarsened crystals. In contrast to chip calorimetry results, large domain sizes are obtained for heating and cooling without isothermal annealing at a conventional rate of 20 K min −1 . This initial morphology then develops further with increased isothermal annealing. The combination of chip calorimetry and ptychography allows separating the effects of each single thermal step on morphology development. [ABSTRACT FROM AUTHOR]

Utilisation of partially coherent X-rays in Coherent X-ray Diffraction Imaging (CDI) and X-ray ptychography have brought intense research and developments for the past few years. Generally, data reconstructions convergence can be achieved by considering to incorporate partially coherent X-rays in ptychographic iterative reconstructions algorithms. Recently, exploration of both X-ray probe and sample modes have been studied and it was suggested that sufficient constraints are required to achieve accurate data reconstructions due to the mathematical inseparability of multiple sample modes that are superimposed into a single set of ptychographic intensity patterns. Here we review the multi-modal ptychography that can be used as a new emerging technique to investigate dynamical sample systems with partially coherent X-rays by recent experimental and simulated demonstrations.

The in situ physicochemical analysis of nanostructured functional materials is crucial for advances in their design and production. X-ray coherent diffraction imaging (CDI) methods have recently demonstrated impressive potential for characterizing such materials with a high spatial resolution and elemental sensitivity; however, moving from the current ex situ static regime to the in situ dynamic one remains a challenge. By combining soft X-ray ptychography and single-shot keyhole CDI, we performed the first in situ spatiotemporal study on an electrodeposition process in a sealed wet environment, employed for the fabrication of oxygen-reduction catalysts, which are key components for alkaline fuel cells and metal-air batteries. The results provide the first experimental demonstration of theoretically predicted Turing-Hopf electrochemical pattern formation resulting from morphochemical coupling, adding a new dimension for the in-depth in situ characterization of electrodeposition processes in space and time. [ABSTRACT FROM AUTHOR]

Holographic and near-field ptychographic X-ray computed tomography techniques have been compared by characterizing a typical solid oxide cell hydrogen electrode using a high-energy X-ray beam. The main advantages and drawbacks of both methods are discussed regarding the possibility to image the Ni-YSZ cermet, a complex porous electrode microstructure composed of X-ray absorbent materials. The same innovative sub-pixel alignment algorithm, based on tomographic consistency, was applied to align the different tomographic projections for each technique. It has been shown that a better signal-to-noise ratio (SNR) is obtained using near-field ptychographic tomography, whereas holographic tomography can be faster with similar spatial resolution. Moreover, quantitative electron density maps have been obtained with the two techniques. The quality of the phase identification has also been assessed and compared in both cases using a classical grey-level class separability criterion. After the segmentation, a set of typical microstructural properties describing the electrode morphology was computed. The comparison of the results allowed validating the complementarity of the two X-ray imaging techniques. Despite the more time-consuming data acquisition and processing than holographic tomography, near-field ptychographic tomography is especially well adapted to image samples without any insight on their composition or when the sample is highly absorbent. Yet, holographic X-ray tomography, using high-energy X-rays to reduce the sample absorption, remains a faster 3D imaging technique with spatial resolution and contrast sensitivity sufficient for the characterization of solid oxide cell materials. • 3D reconstruction of a Solid Oxide Cell electrode synchrotron X-ray holography and near-field ptychography using hard X-rays. • Comparison of the two reconstruction techniques in terms of acquisition time, computational intensity, quality of the 3D images. • Quantitative electron density 3D maps obtained for the sample with the two methods • Validation of the the microstructural parameters extracted from the raw 3D reconstructions. [ABSTRACT FROM AUTHOR]

We study the assembly of DNA-functionalized nanocubes under lateral confinement in microscale square trenches on a DNA-functionalized substrate. Microfocus small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) are used to characterize the superlattices (SLs). The results indicate that nanocubes form simple-cubic SLs with square-prism morphology and a (100) out-of-plane orientation to maximize DNA bonding. In-plane, SLs align with the template, exposing their {100} side facets, and the degree of alignment depends on trench size. Interestingly, the distribution of in-plane orientations determined from SAXS and SEM do not agree, indicating that the internal and external structures of the SLs differ. To understand this discrepancy, X-ray ptychography is employed to image the internal structures of the SLs, revealing that SLs which appear to be single-crystalline in SEM may have subsurface grain boundaries, depending on trench size. SEM reveals that the SLs grow via nucleation and growth of randomly oriented domains, which then coalesce; this mechanism explains the observed dependence of alignment and defect structure on size. Interestingly, crystallization occurs via an unusual growth mode, whereby continuous SL layers grow on top of several misoriented islands. Overall, this work elucidates the effect of lateral confinement on the crystallization of DNA-functionalized nanoparticles and shows how X-ray ptychography can be used to gain insight into nanoparticle crystallization.