Current status of the TwinMic beamline at Elettra: a soft X‐ray transmission and emission microscopy station

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X‐ray microscopy station, is reported. The X‐ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy‐tailored using a collimated plane‐grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full‐field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X‐ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across‐absorption‐edge imaging, X‐ray absorption near‐edge structure or low‐energy X‐ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results.     

Dose,exposure time and resolution in serial X‐ray crystallography

The resolution of X‐ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed serial crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single‐file train across a continuous X‐ray beam, and resolution is then limited only by the available molecular and X‐ray fluxes and molecular alignment. Orientation of the diffracting molecules is achieved by laser alignment. The incident X‐ray fluence (energy/area) is evaluated that is required to obtain a given resolution from (i) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (ii) explicit simulation of diffraction patterns for a GroEL–GroES protein complex, and (iii) the spatial frequency cut‐off of the transfer function following iterative solution of the phase problem, and reconstruction of an electron density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate counting time and the number of proteins needed within the beam at any instant for a given resolution and X‐ray flux. An inverse fourth‐power dependence of exposure time on resolution is confirmed, with important implications for all coherent X‐ray imaging. It is found that multiple single‐file protein beams will be needed for sub‐nanometer resolution on current third‐generation synchrotrons, but not on fourth‐generation designs, where reconstruction of secondary protein structure at a resolution of 7 Å should be possible with relatively short exposures.     

Three‐dimensional imaging of chemical phase transformations at the nanoscale with full‐field transmission X‐ray microscopy

The ability to probe morphology and phase distribution in complex systems at multiple length scales unravels the interplay of nano‐ and micrometer‐scale factors at the origin of macroscopic behavior. While different electron‐ and X‐ray‐based imaging techniques can be combined with spectroscopy at high resolutions, owing to experimental time limitations the resulting fields of view are too small to be representative of a composite sample. Here a new X‐ray imaging set‐up is proposed, combining full‐field transmission X‐ray microscopy (TXM) with X‐ray absorption near‐edge structure (XANES) spectroscopy to follow two‐dimensional and three‐dimensional morphological and chemical changes in large volumes at high resolution (tens of nanometers). TXM XANES imaging offers chemical speciation at the nanoscale in thick samples (>20 µm) with minimal preparation requirements. Further, its high throughput allows the analysis of large areas (up to millimeters) in minutes to a few hours. Proof of concept is provided using battery electrodes, although its versatility will lead to impact in a number of diverse research fields.     

A sub‐micrometer resolution hard X‐ray microprobe system of BL8C at Pohang Light Source

A microprobe system has been installed on the nanoprobe/XAFS beamline (BL8C) at PLS‐II, South Korea. Owing to the reproducible switch of the gap of the in‐vacuum undulator (IVU), the intense and brilliant hard X‐ray beam of an IVU can be used in X‐ray fluorescence (XRF) and X‐ray absorption fine‐structure (XAFS) experiments. For high‐spatial‐resolution microprobe experiments a Kirkpatrick–Baez mirror system has been used to focus the millimeter‐sized X‐ray beam to a micrometer‐sized beam. The performance of this system was examined by a combination of micro‐XRF imaging and micro‐XAFS of a beetle wing. These results indicate that the microprobe system of the BL8C can be used to obtain the distributions of trace elements and chemical and structural information of complex materials.     

Utilizing broadband X‐rays in a Bragg coherent X‐ray diffraction imaging experiment

A method is presented to simplify Bragg coherent X‐ray diffraction imaging studies of complex heterogeneous crystalline materials with a two‐stage screening/imaging process that utilizes polychromatic and monochromatic coherent X‐rays and is compatible with in situ sample environments. Coherent white‐beam diffraction is used to identify an individual crystal particle or grain that displays desired properties within a larger population. A three‐dimensional reciprocal‐space map suitable for diffraction imaging is then measured for the Bragg peak of interest using a monochromatic beam energy scan that requires no sample motion, thus simplifying in situ chamber design. This approach was demonstrated with Au nanoparticles and will enable, for example, individual grains in a polycrystalline material of specific orientation to be selected, then imaged in three dimensions while under load.     

High‐resolution thermal imaging with a combination of nano‐focus X‐ray diffraction and ultra‐fast chip calorimetry

A microelectromechanical‐systems‐based calorimeter designed for use on a synchrotron nano‐focused X‐ray beamline is described. This instrument allows quantitative DC and AC calorimetric measurements over a broad range of heating/cooling rates (≤100000 K s^?1) and temperature modulation frequencies (≤1 kHz). The calorimeter was used for high‐resolution thermal imaging of nanogram‐sized samples subjected to X‐ray‐induced heating. For a 46 ng indium particle, the measured temperature rise reaches ～0.2 K, and is directly correlated to the X‐ray absorption. Thermal imaging can be useful for studies of heterogeneous materials exhibiting physical and/or chemical transformations. Moreover, the technique can be extended to three‐dimensional thermal nanotomography.     

X‐ray spectromicroscopy in soil and environmental sciences

X‐ray microscopy is capable of imaging particles in the nanometer size range directly with sub‐micrometer spatial resolution and can be combined with high spectral resolution for spectromicroscopy studies. Two types of microscopes are common in X‐ray microscopy: the transmission X‐ray microscope and the scanning transmission X‐ray microscope; their set‐ups are explained in this paper. While the former takes high‐resolution images from an object with exposure times of seconds or faster, the latter is very well suited as an analytical instrument for spectromicroscopy. The morphology of clusters or particles from soil and sediment samples has been visualized using a transmission X‐ray microscope. Images are shown from a cryo‐tomography experiment based on X‐ray microscopy images to obtain information about the three‐dimensional structure of clusters of humic substances. The analysis of a stack of images taken with a scanning transmission X‐ray microscope to combine morphology and chemistry within a soil sample is shown. X‐ray fluorescence is a method ideally applicable to the study of elemental distributions and binding states of elements even on a trace level using X‐ray energies above 1 keV.     

Coherent X‐ray scattering beamline at port 9C of Pohang Light Source II

The coherent X‐ray scattering beamline at the 9C port of the upgraded Pohang Light Source (PLS‐II) at Pohang Accelerator Laboratory in Korea is introduced. This beamline provides X‐rays of 5–20 keV, and targets coherent X‐ray experiments such as coherent diffraction imaging and X‐ray photon correlation spectroscopy. The main parameters of the beamline are summarized, and some preliminary experimental results are described.     

Full‐field X‐ray reflection microscopy of epitaxial thin‐films

Novel X‐ray imaging of structural domains in a ferroelectric epitaxial thin film using diffraction contrast is presented. The full‐field hard X‐ray microscope uses the surface scattering signal, in a reflectivity or diffraction experiment, to spatially resolve the local structure with 70 nm lateral spatial resolution and sub‐nanometer height sensitivity. Sub‐second X‐ray exposures can be used to acquire a 14 µm × 14 µm image with an effective pixel size of 20 nm on the sample. The optical configuration and various engineering considerations that are necessary to achieve optimal imaging resolution and contrast in this type of microscopy are discussed.     

Optimization of analyzer‐based imaging systems for minimal surface absorbed dose

Analyzer‐based imaging has improved tissue X‐ray imaging beyond what conventional radiography was able to achieve. The extent of the improvement is dependent on the crystal reflection used in the monochromator and analyzer combination, the imaging photon energy, the geometry of the sample and the imaging detector. These many factors determine the ability of the system to distinguish between various bone tissues or soft tissues with a specified statistical certainty between pixels in a counting detector before any image processing. The following discussion will detail changes in the required number of imaging photons and the resulting surface absorbed dose when the imaging variables are altered. The process whereby the optimal imaging parameters to deliver the minimum surface absorbed dose to a sample while obtaining a desired statistical certainty between sample materials for an arbitrary analyzer‐based imaging system will be described. Two‐component samples consisting of bone and soft tissue are discussed as an imaging test case. The two‐component approach will then be generalized for a multiple‐component sample.     

Optical performance of materials for X‐ray refractive optics in the energy range 8–100 keV

A quantitative analysis of the crucial characteristics of currently used and promising materials for X‐ray refractive optics is performed in the extended energy range 8–100 keV. According to the examined parameters, beryllium is the material of choice for X‐ray compound refractive lenses (CRLs) in the energy range 8–25 keV. At higher energies the use of CRLs made of diamond and the cubic phase of boron nitride (c‐BN) is beneficial. It was demonstrated that the presence of the elements of the fourth (or higher) period has a fatal effect on the functional X‐ray properties even if low‐Z elements dominate in the compound, like in YB₆₆. Macroscopic properties are discussed: much higher melting points and thermal conductivities of C and c‐BN enable them to be used at the new generation of synchrotron radiation sources and X‐ray free‐electron lasers. The role of crystal and internal structure is discussed: materials with high density are preferable for refractive applications while less dense phases are suitable for X‐ray windows. Single‐crystal or amorphous glass‐like materials based on Li, Be, B or C that are free of diffuse scattering from grain boundaries, voids and inclusions are the best candidates for applications of highly coherent X‐ray beams.     

Spectrometer for hard X‐ray free‐electron laser based on diffraction focusing

X‐ray free‐electron lasers (XFELs) generate sequences of ultra‐short spatially coherent pulses of X‐ray radiation. A diffraction focusing spectrometer (DFS), which is able to measure the whole energy spectrum of the radiation of a single XFEL pulse with an energy resolution of ΔE/E? 2 × 10^?6, is proposed. This is much better than for most modern X‐ray spectrometers. Such resolution allows one to resolve the fine spectral structure of the XFEL pulse. The effect of diffraction focusing occurs in a single‐crystal plate due to dynamical scattering, and is similar to focusing in a Pendry lens made from a metamaterial with a negative refraction index. Such a spectrometer is easier to operate than those based on bent crystals. It is shown that the DFS can be used in a wide energy range from 5 keV to 20 keV.     

Simultaneous small‐ and wide‐angle scattering at high X‐ray energies

Combined small‐ and wide‐angle X‐ray scattering (SAXS/WAXS) is a powerful technique for the study of materials at length scales ranging from atomic/molecular sizes (a few angstroms) to the mesoscopic regime (～1 nm to ～1 µm). A set‐up to apply this technique at high X‐ray energies (E > 50 keV) has been developed. Hard X‐rays permit the execution of at least three classes of investigations that are significantly more difficult to perform at standard X‐ray energies (8–20 keV): (i) in situ strain analysis revealing anisotropic strain behaviour both at the atomic (WAXS) as well as at the mesoscopic (SAXS) length scales, (ii) acquisition of WAXS patterns to very large q (>20 Å^?1) thus allowing atomic pair distribution function analysis (SAXS/PDF) of micro‐ and nano‐structured materials, and (iii) utilization of complex sample environments involving thick X‐ray windows and/or samples that can be penetrated only by high‐energy X‐rays. Using the reported set‐up a time resolution of approximately two seconds was demonstrated. It is planned to further improve this time resolution in the near future.     

Brownian and advective dynamics in microflow studied by coherent X‐ray scattering experiments

Combining microfluidics with coherent X‐ray illumination offers the possibility to not only measure the structure but also the dynamics of flowing samples in a single‐scattering experiment. Here, the power of this combination is demonstrated by studying the advective and Brownian dynamics of colloidal suspensions in microflow of different geometries. Using an experimental setup with a fast two‐dimensional detector and performing X‐ray correlation spectroscopy by calculating two‐dimensional maps of the intensity auto‐correlation functions, it was possible to evaluate the sample structure and furthermore to characterize the detailed flow behavior, including flow geometry, main flow directions, advective flow velocities and diffusive dynamics. By scanning a microfocused X‐ray beam over a microfluidic device, the anisotropic auto‐correlation functions of driven colloidal suspensions in straight, curved and constricted microchannels were mapped with the spatial resolution of the X‐ray beam. This method has not only a huge potential for studying flow patterns in complex fluids but also to generally characterize anisotropic dynamics in materials.     

Synchrotron‐radiation phase‐contrast imaging of human stomach and gastric cancer: in vitro studies

The electron density resolution of synchrotron‐radiation phase‐contrast imaging (SR‐PCI) is 1000 times higher than that of conventional X‐ray absorption imaging in light elements, through which high‐resolution X‐ray imaging of biological soft tissue can be achieved. For biological soft tissue, SR‐PCI can give better imaging contrast than conventional X‐ray absorption imaging. In this study, human resected stomach and gastric cancer were investigated using in‐line holography and diffraction enhanced imaging at beamline 4W1A of the Beijing Synchrotron Radiation Facility. It was possible to depict gastric pits, measuring 50–70 µm, gastric grooves and tiny blood vessels in the submucosa layer by SR‐PCI. The fine structure of a cancerous ulcer was displayed clearly on imaging the mucosa. The delamination of the gastric wall and infiltration of cancer in the submucosa layer were also demonstrated on cross‐sectional imaging. In conclusion, SR‐PCI can demonstrate the subtle structures of stomach and gastric cancer that cannot be detected by conventional X‐ray absorption imaging, which prompt the X‐ray diagnosis of gastric disease to the level of the gastric pit, and has the potential to provide new methods for the imageology of gastric cancer.     

Multimodal hard X‐ray imaging of a mammography phantom at a compact synchrotron light source

The Compact Light Source is a miniature synchrotron producing X‐rays at the interaction point of a counter‐propagating laser pulse and electron bunch through the process of inverse Compton scattering. The small transverse size of the luminous region yields a highly coherent beam with an angular divergence of a few milliradians. The intrinsic monochromaticity and coherence of the produced X‐rays can be exploited in high‐sensitivity differential phase‐contrast imaging with a grating‐based interferometer. Here, the first multimodal X‐ray imaging experiments at the Compact Light Source at a clinically compatible X‐ray energy of 21 keV are reported. Dose‐compatible measurements of a mammography phantom clearly demonstrate an increase in contrast attainable through differential phase and dark‐field imaging over conventional attenuation‐based projections.     

In‐line Bragg magnifier based on V‐shaped germanium crystals

In this work an X‐ray imaging system based on a recently developed in‐line two‐dimensional Bragg magnifier composed of two monolithic V‐shaped crystals made of dislocation‐free germanium is presented. The channel‐cut crystals were used in one‐dimensional and in two‐dimensional (crossed) configurations in imaging applications and allowed measurement of phase‐contrast radiograms both in the edge‐enhanced and in the holographic regimes. The measurement of the phase gradient in two orthogonal directions is demonstrated. The effective pixel size attained was 0.17 µm in the one‐dimensional configuration and 0.5 µm in the two‐dimensional setting, offering a twofold improvement in spatial resolution over devices based on silicon. These results show the potential for applying Bragg magnifiers to imaging soft matter at high resolution with reduced dose owing to the higher efficiency of Ge compared with Si.     

Single‐cell resolution in high‐resolution synchrotron X‐ray CT imaging with gold nanoparticles

Gold nanoparticles are excellent intracellular markers in X‐ray imaging. Having shown previously the suitability of gold nanoparticles to detect small groups of cells with the synchrotron‐based computed tomography (CT) technique both ex vivo and in vivo, it is now demonstrated that even single‐cell resolution can be obtained in the brain at least ex vivo. Working in a small animal model of malignant brain tumour, the image quality obtained with different imaging modalities was compared. To generate the brain tumour, 1 × 10⁵ C6 glioma cells were loaded with gold nanoparticles and implanted in the right cerebral hemisphere of an adult rat. Raw data were acquired with absorption X‐ray CT followed by a local tomography technique based on synchrotron X‐ray absorption yielding single‐cell resolution. The reconstructed synchrotron X‐ray images were compared with images obtained by small animal magnetic resonance imaging. The presence of gold nanoparticles in the tumour tissue was verified in histological sections.     

Ultrafast switching of hard X‐rays

A new concept for shortening hard X‐ray pulses emitted from a third‐generation synchrotron source down to few picoseconds is presented. The device, called the PicoSwitch, exploits the dynamics of coherent acoustic phonons in a photo‐excited thin film. A characterization of the structure demonstrates switching times of ≤ 5 ps and a peak reflectivity of ～10^?3. The device is tested in a real synchrotron‐based pump–probe experiment and reveals features of coherent phonon propagation in a second thin film sample, thus demonstrating the potential to significantly improve the temporal resolution at existing synchrotron facilities.     

A multiplexed high‐resolution imaging spectrometer for resonant inelastic soft X‐ray scattering spectroscopy

The optical design of a two‐dimensional imaging soft X‐ray spectrometer is described. A monochromator will produce a dispersed spectrum in a narrow vertical illuminated stripe (～2 µm wide by ～2 mm tall) on a sample. The spectrometer will use inelastically scattered X‐rays to image the extended field on the sample in the incident photon energy direction (vertical), resolving the incident photon energy. At the same time it will image and disperse the scattered photons in the orthogonal (horizontal) direction, resolving the scattered photon energy. The principal challenge is to design a system that images from the flat‐field illumination of the sample to the flat field of the detector and to achieve sufficiently high spectral resolution. This spectrometer provides a completely parallel resonant inelastic X‐ray scattering measurement at high spectral resolution (～30000) over the energy bandwidth (～5 eV) of a soft X‐ray absorption resonance.