Transmission X-ray microscopes (TXMs) are now increasingly used for quantitative analysis of samples, most notably in the spectral analysis of materials. Validating such measurements requires quantitatively accurate models for these microscopes, but current TXM models have only been tested qualitatively. Here we develop an experimental and theoretical framework for evaluation of TXMs that uses Mie theory to compute the electric field emerging from a nanosphere. We approximate the microscope’s condenser illumination by plane waves at the mean illumination angle and the zone plate by a thin lens. We find that this model produces good qualitative agreement with our 3D measurements of 60 nm gold nanospheres, but only if both β and δ for the complex refractive index n = 1 – δ + iβ of gold are included in the model. This shows that both absorption and phase properties of the specimen influence the acquired TXM image. The qualitative agreement improves if we incorporate a small tilt into the condenser illumination relative to the optical axis, implying a small misalignment in the microscope. Finally, in quantitative comparisons, we show that the model predicts the nanosphere’s expected absorption as determined by Beer’s law, whereas the microscope underestimates this absorption by 10–20%. This surprising observation highlights the need for future work to identify the microscope feature(s) that lead to this quantitative discrepancy.

Modern soft X-ray beamlines use collimated plane-grating monochromators (cPGM) [1] as monochromatization devices. Multilayer (ML)-coated plane gratings and mirrors allow to extend the available photon energy range of cPGMs towards the so-called tender X-ray photon energy range (up to 5keV), thereby achieving a significantly higher photon flux [2]. This X-ray energy range covers the L- and M-absorption edges of most transition and rare earth metals as well as the K-edges of lighter elements such as silicon, sulfur and phosphorus. Recently, such an ML-based monochromator setup was put into operation at the beamline U41-PGM1-XM at the storage ring BESSY-II in Berlin [3]. This beamline upgrade enables high-resolution spectromicroscopic applications with photon energies of up to 3keV and it is expanding its capabilities to support research in areas such as life sciences, semiconductor development and battery research. We report on the construction, commissioning and performance of this beamline and discuss possible options for new developments in the area of beamlines and end-stations in the tender X-ray energy range (up to 5keV) at existing and future new accelerator-based photon sources.

In this study, a novel application of synchrotron X-ray nanotomography based on high-resolution full-field transmission X-ray microscopy for characterizing the structure and morphology of micrometric hollow polymeric fibers is presented. By employing postimage analysis using an open-source software such as Tomviz and ImageJ, various key parameters in fiber morphology, including diameter, wall thickness, wall thickness distribution, pore size, porosity, and surface roughness, were assessed. Electrospun polycaprolactone fibers with micrometric diameters and submicrometric features with induced porosity via gas dissolution foaming were used to this aim. The acquired synchrotron X-ray nanotomography data were analyzed using two approaches: 3D tomographic reconstruction and 2D radiographic projection-based analysis. The results of the combination of both approaches demonstrate unique capabilities of this technique, not achievable by other available techniques, allowing for a full characterization of the internal and external morphology and structure of the fibers as well as to obtain valuable qualitative insights into the overall fiber structure.

Layered lithium-rich transition metal oxides are promising cathode candidates for high-energy-density lithium batteries due to the redox contributions from transition metal cations and oxygen anions. However, their practical application is hindered by gradual capacity fading and voltage decay. Although oxygen loss and phase transformation are recognized as primary factors, the structural deterioration, chemical rearrangement, kinetic and thermodynamic effects remain unclear. Here we integrate analysis of morphological, structural and oxidation state evolution from individual atoms to secondary particles. By performing nanoscale to microscale characterizations, distinct structural change pathways associated with intraparticle heterogeneous reactions are identified. The high level of oxygen defects formed throughout the particle by slow electrochemical activation triggers progressive phase transformation and the formation of nanovoids. Ultrafast lithium (de)intercalation leads to oxygen-distortion-dominated lattice displacement, transition metal ion dissolution and lithium site variation. These inhomogeneous and irreversible structural changes are responsible for the low initial Coulombic efficiency, and ongoing particle cracking and expansion in the subsequent cycles. This work employs nano- to microscale characterization to identify different structural change pathways associated with non-homogeneous reactions within the particles, and explores differences in the failure mechanisms of lithium-rich transition metal oxide materials at different current densities.

In this paper, we give an overview of the nanoscale spectromicroscopy studies performed with the full-field X-ray microscope at the BESSY II electron storage ring. We do not consider spectromicroscopy studies performed with X-ray microscopes operated at other synchrotron sources. Such studies can be found in the literature. To our knowledge, the full-field X-ray microscope at the BESSY II storage ring is the first one operating with both a plane-grating-monochromator (PGM) beamline equipped with multi-layer optics for the tender X-ray range, as well as with standard optical elements for the soft X-ray range. We discuss how this instrument has been used in various published NEXAFS-TXM studies to probe low dimensional nanostructures. This research work paves the way for understanding electronic structures approaching the atomic scale, and will thereby help in the design of tailored functional systems.

The 3D morphology of hierarchically structured electrocatalytic systems is determined based on multi-scale X-ray computed tomography (XCT), and the crystalline structure of electrocatalyst nanoparticles is characterized using transmission electron microscopy (TEM), supported by X-ray diffraction (XRD) and spatially resolved near-edge X-ray absorption fine structure (NEXAFS) studies. The high electrocatalytic efficiency for hydrogen evolution reaction (HER) of a novel transition-metal-based material system - MoNi4 electrocatalysts anchored on MoO2 cuboids aligned on Ni foam (MoNi4/MoO2@Ni) - is based on advantageous crystalline structures and chemical bonding. High-resolution TEM images and selected-area electron diffraction patterns are used to determine the crystalline structures of MoO2 and MoNi4. Multi-scale XCT provides 3D information of the hierarchical morphology of the MoNi4/MoO2@Ni material system nondestructively: Micro-XCT images clearly resolve the Ni foam and the attached needle-like MoO2 micro cuboids. Laboratory nano-XCT shows that the MoO2 micro cuboids with a rectangular cross-section of 0.5 × 1 µm2 and a length of 10-20 µm are vertically arranged on the Ni foam. MoNi4 nanoparticles with a size of 20-100 nm, positioned on single MoO2 cuboids, were imaged using synchrotron radiation nano-XCT. The application of a deep convolutional neural network (CNN) significantly improves the reconstruction quality of the acquired data.

The combination of near edge X-ray absorption spectroscopy with nanoscale X-ray imaging is a powerful analytical tool for many applications in energy technologies, catalysis, which are critical to combat climate change, as well as microelectronics and life science. Materials from these scientific areas often contain key elements, such as Si, P, S, Y, Zr, Nb, and Mo as well as lanthanides, whose X-ray absorption edges lie in the so-called tender photon energy range 1.5-5.0 keV. Neither conventional grazing incidence grating nor crystal monochromators have high transmission in this energy range, thereby yielding the tender photon energy gap. To close this gap, a monochromator setup based on a multilayer coated blazed plane grating and plane mirror is devised. The measurements show that this novel concept improves the photon flux in the tender X-ray regime by two-orders-of-magnitude enabling previously unattainable laboratory and synchrotron-based studies. This setup is applied to perform nanoscale spectromicroscopy studies. The high photon flux provides sufficient sensitivity to obtain the electronic structure of Mo in platinum-free MoNi4 nanoparticles for electrochemical energy conversion. Additionally, it is shown that the chemical bonding of nano-structures in integrated circuits can be distinguished by the electronic configuration at the Si-K edge.

Holography is a powerful method for achieving 3D images of objects. Extending this method to short wavelengths potentially offers significantly higher resolution than visible light holography. However, current X-ray holography setups employ nanoscale pinholes to form the reference wave. This approach is relatively inefficient and limited to very small sample size. Here, we propose a new setup for X-ray holography based on a binary diffractive optical element (DOE), which forms at the same time the object illumination and the reference wave. This optic is located separately from the sample plane, which permits investigation of larger sample areas. Using an extended test sample, we demonstrate a resolution of 90 nm (half-pitch) at an undulator beamline at BESSY II. The new holography setup can be directly transferred to free electron laser sources enabling time-resolved nanoscale imaging for ultra-fast processes.

TiO2 nanoparticles doped with different amounts of Nd3+ (0.5, 1, and 3 wt.%) were synthetized by the sol–gel method, and evaluated as potential temperature nanoprobes using the fluorescence intensity ratio between thermal-sensitive radiative transitions of the Nd3+. XRD characterization identified the anatase phase in all the doped samples. The morphology of the nanoparticles was observed with SEM, TEM and HRTEM microscopies. The relative amount of Nd3+ in TiO2 was obtained by EDXS, and the oxidation state of titanium and neodymium was investigated via XPS and NEXAFS, respectively. Nd3+ was present in all the samples, unlike titanium, where besides Ti4+, a significantly amount of Ti3+ was observed; the relative concentration of Ti3+ increased as the amount of Nd3+ in the TiO2 nanoparticles increased. The photoluminescence of the synthetized nanoparticles was investigated, with excitation wavelengths of 350, 514 and 600 nm. The emission intensity of the broad band that was associated with the presence of defects in the TiO2, increased when the concentration of Nd3+ was increased. Using 600 nm for excitation, the 4F7/2→4I9/2, 4F5/2→4I9/2 and 4F3/2→4I9/2 transitions of Nd3+ ions, centered at 760 nm, 821 nm, and 880 nm, respectively, were observed. Finally, the effect of temperature in the photoluminescence intensity of the synthetized nanoparticles was investigated, with an excitation wavelength of 600 nm. The spectra were collected in the 288–348 K range. For increasing temperatures, the emission intensity of the 4F7/2→4I9/2 and 4F5/2→4I9/2 transitions increased significantly, in contrast to the 4F3/2→4I9/2 transition, in which the intensity emission decreased. The fluorescence intensity ratio between the transitions I821I880=F5/24I49/2F43/2I49/2 and I760I880=F47/2I49/2F43/2I49/2 were used to calculate the relative sensitivity of the sensors. The relative sensitivity was near 3% K−1 for I760I880 and near 1% K−1 for I821I880.

The diatom shell is an example of complex siliceous structure which is a suitable model to demonstrate the process of digging into the third dimension using modern visualization techniques. This paper demonstrates importance of a comprehensive multi-length scale approach to the bio-structures/materials with the usage of state-of-the-art imaging techniques. Imaging of diatoms applying visible light, electron and X-ray microscopy provide a deeper insight into the morphology of their frustules.

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.

AbstractTemperature is a critical parameter in biology, affecting the speed of reactions that occur in living systems. Nevertheless, measuring temperature with subcellular resolution (micrometric scale) and reliability remains a challenge to overcome. In this perspective, luminescence nanothermometry is a non‐contact technique which aims to measure temperature with a sub‐micrometric spatial resolution through the use of nanomaterials whose luminescence is affected solely by changes in temperature. Here, TiO2 nanoparticles doped with Eu+3 ions (Eu+3‐TiO2) are used for sensing temperature differences within single living cells. XRD, XPS, SEM, TEM and NEXAFS analysis allow the determination of the physicochemical characteristics of the Eu+3‐TiO2 nanoparticles and, the variation of the luminescence intensity of the Eu+3‐TiO2 nanoparticles with their temperature is investigated. The successful internalization of Eu+3‐TiO2 nanoparticles in different types of cells is observed. The luminescence of nanoparticles internalized in L929 fibroblast cells is measured when the system is heated in a biological relevant temperature range. Making use of an appropriate calibration curve the temperature variation inside the cells is determined with sensitivity of 0.5 K per 1% of luminosity change when heated.

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2's known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.

Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLSfunctionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.

To map morphological changes of circulating monocytes between CSF1R-related leukoencephalopathy and healthy donors using nano x-ray tomography and electron microscope, with a specific focus on the numbers and volumes of secondary lysosomes.

Here we use cryo soft X-ray tomography (cryo SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insights about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), by cryo SXT we detected ~5% of dPGS-AuNP in the cytoplasm, a level undetectable by con-focal light microscopy. We also observed ~5% of dPGS-AuNP in a rarely identified sub-cellular site, namely lipid drop-lets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1-6 h incubation period; the number of small vesicles and mitochondria significantly increased and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethyleneimine (PEI), used for nucleic acid delivery. PEI nanopar-ticles did not enter lipid droplets, but they induced similar, albeit less pronounced changes in the quantity of cyto-plasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.

N-doped TiO2 materials have recently attracted a considerable amount of interest due to their enhanced photoelectrochemical properties compared to pristine TiO2. However, this improvement is still not clearly correlated to the N chemistry because the attribution of the observed components in the N is X-ray photoelectron spectra is strongly disputed. In this context, this joint experimental and theoretical study aims at clearly distinguishing the different N atomic species and positions that can be encountered upon N-doping of titania by the N-2(+) ion implantation method. Core-level binding energy shifts (CLS) calculated at a quantum-chemical level for N is orbitals with different chemical environments nicely match the components obtained by the fitting of the N is XPS experimental regions and allow for its detailed interpretation. We also monitored the N atom positions as a function of the dose used for implantation. Based on classical simulations of ion/surface interactions achieved with the help of the TRIDYN software, we show that oxygen vacancies play a key role in defining the nitrogen doping type (i.e., nitrogen position in the TiO2 lattice), as further supported by DFT calculations.

Combined proteomics, nanotomography, and live cell spectroscopy help relate gold nanoparticle processing to the interactions at the nanoparticle interface.