Since 2013, three beamlines for macromolecular crystallography are available to users at the thirdgeneration synchrotron PETRA III in Hamburg: P11, P13 and P14, the latter two operated by EMBL. Beamline P11 is operated by DESY and is equipped with a Pilatus 6M detector. Together with the photon flux of 2 × 10 13 ph/s provided by the very brilliant X-ray source of PETRA III, a full data set can be typically collected in less than 2 min. P11 provides state-of-the-art microfocusing capabilities with beam sizes down to 1 × 1 µm 2 , which makes the beamline ideally suited for investigation of microcrystals and serial crystallography experiments. An automatic sample changer allows fast sample exchange in less than 20 s, which enables high-throughput crystallography and fast crystal screening. For sample preparation, an S2 biosafety laboratory is available in close proximity to the beamline.

Multilayer Laue lenses are volume diffraction elements for the efficient focusing of X-rays. With a new manufacturing technique that we introduced, it is possible to fabricate lenses of sufficiently high numerical aperture (NA) to achieve focal spot sizes below 10 nm. The alternating layers of the materials that form the lens must span a broad range of thicknesses on the nanometer scale to achieve the necessary range of X-ray deflection angles required to achieve a high NA. This poses a challenge to both the accuracy of the deposition process and the control of the materials properties, which often vary with layer thickness. We introduced a new pair of materials—tungsten carbide and silicon carbide—to prepare layered structures with smooth and sharp interfaces and with no material phase transitions that hampered the manufacture of previous lenses. Using a pair of multilayer Laue lenses (MLLs) fabricated from this system, we achieved a two-dimensional focus of 8.4 × 6.8 nm2 at a photon energy of 16.3 keV with high diffraction efficiency and demonstrated scanning-based imaging of samples with a resolution well below 10 nm. The high NA also allowed projection holographic imaging with strong phase contrast over a large range of magnifications. An error analysis indicates the possibility of achieving 1 nm focusing.

The ever-increasing brightness of synchrotron radiation sources demands improved X-ray optics to utilize their capability for imaging and probing biological cells, nano-devices and functional matter on the nanometre scale with chemical sensitivity. Hard X-rays are ideal for high-resolution imaging and spectroscopic applications owing to their short wavelength, high penetrating power and chemical sensitivity. The penetrating power that makes X-rays useful for imaging also makes focusing them technologically challenging. Recent developments in layer deposition techniques have enabled the fabrication of a series of highly focusing X-ray lenses, known as wedged multi-layer Laue lenses. Improvements to the lens design and fabrication technique demand an accurate, robust, in situ and at-wavelength characterization method. To this end, a modified form of the speckle tracking wavefront metrology method has been developed. The ptychographic X-ray speckle tracking method is capable of operating with highly divergent wavefields. A useful by-product of this method is that it also provides high-resolution and aberration-free projection images of extended specimens. Three separate experiments using this method are reported, where the ray path angles have been resolved to within 4 nrad with an imaging resolution of 45 nm (full period). This method does not require a high degree of coherence, making it suitable for laboratory-based X-ray sources. Likewise, it is robust to errors in the registered sample positions, making it suitable for X-ray free-electron laser facilities, where beam-pointing fluctuations can be problematic for wavefront metrology.

X-ray ptychography is an ultrahigh-resolution scanning coherent diffractive imaging technique, allowing quantitative measurements of extended samples and a simultaneous reconstruction of the illuminating wavefront. Recent development of the mixed-state reconstruction algorithm has triggered a certain interest in utilizing partially coherent X-ray sources for ptychography. Here, we study how finite spatial coherence influences the reconstructed image of a test structure. Our work shows that use of a highly coherent illumination provides images with better spatial resolution and fewer artefacts than the approach with partial coherence.

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacteriumtuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychographyenhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix. Metal ions play an important role in the vital functions of living organisms. They are present in various biological systems in a vast range of concentrations as structural, electrolyte (minor), and trace elements. From being major tissue components (e.g. Ca in bones) to constituents of essential biological molecules (e.g. Fe in hemoglobin), metals take part in the majority of extra-and intracellular processes. In particular, first-row transition metals (Mn, Fe, Cu, Ni, Zn)-despite their minute concentrations-are involved in sub-cellular processes. Moreover, their abnormal accumulation in human brain was correlated with mechanisms leading to neurodegeneration diseases such as Parkinson's and Alzheimer's 1-3. Metal compounds have also been utilized as novel drug delivery systems by addressing metabolic properties of bacterial agents 4,5 or as more efficient medical imaging markers tracking tissue of interest 6-8. In all aforementioned cases, studies of metal contributions require knowledge of their quantitative spatial distributions with respect to the sub-cellular structure. Electron-probe Energy-Dispersive Spectroscopy offers elemental mapping at nanometer-range spatial resolutions and high excitation efficiency in the low-Z-element