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Nature Communications 11(1), 4511 (2020). doi:10.1038/s41467-020-18156-7, Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported., Published by Nature Publishing Group UK, [London]

IUCrJ 6(3), 1-9 (2019). doi:10.1107/S2052252519002781, The routine atomic resolution structure determination of single particles is expected to have profound implications for probing structure–function relationships in systems ranging from energy-storage materials to biological molecules. Extremely bright ultrashort-pulse X-ray sources – X-ray free-electron lasers (XFELs) – provide X-rays that can be used to probe ensembles of nearly identical nanoscale particles. When combined with coherent diffractive imaging, these objects can be imaged; however, as the resolution of the images approaches the atomic scale, the measured data are increasingly difficult to obtain and, during an X-ray pulse, the number of photons incident on the 2D detector is much smaller than the number of pixels. This latter concern, the signal `sparsity', materially impedes the application of the method. An experimental analog using a conventional X-ray source is demonstrated and yields signal levels comparable with those expected from single biomolecules illuminated by focused XFEL pulses. The analog experiment provides an invaluable cross check on the fidelity of the reconstructed data that is not available during XFEL experiments. Using these experimental data, it is established that a sparsity of order 1.3 × 10−3 photons per pixel per frame can be overcome, lending vital insight to the solution of the atomic resolution XFEL single-particle imaging problem by experimentally demonstrating 3D coherent diffractive imaging from photon-sparse random projections., Published by Chester

Optica 8(1), 15 - 23 (2021). doi:10.1364/OPTICA.410851, Single particle imaging at x-ray free electron lasers (XFELs) has the potential to determine the structure and dynamics of single biomolecules at room temperature. Two major hurdles have prevented this potential from being reached, namely, the collection of sufficient high-quality diffraction patterns and robust computational purification to overcome structural heterogeneity. We report the breaking of both of these barriers using gold nanoparticle test samples, recording around 10 million diffraction patterns at the European XFEL and structurally and orientationally sorting the patterns to obtain better than 3-nm-resolution 3D reconstructions for each of four samples. With these new developments, integrating advancements in x-ray sources, fast-framing detectors, efficient sample delivery, and data analysis algorithms, we illuminate the path towards sub-nanometer biomolecular imaging. The methods developed here can also be extended to characterize ensembles that are inherently diverse to obtain their full structural landscape., Published by OSA, Washington, DC

Communications Physics 3(1), 97 (2020). doi:10.1038/s42005-020-0362-y, The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate., Published by Springer Nature, London

The free-electron laser FLASH2, a variable gap undulators line, has opened new scientific possibilities for users at DESY in the Hamburg area [1]. The current pulsed radiation at the FLASH facility primarily relies on the SASE process. Thus, the beam characteristics may differ drastically from pulse to pulse; requiring single-shot photon diagnostics and characterization of the photon beam parameters. The beamline FL24 at FLASH2 is equipped with a set of bendable Kirkpatrick-Baez (KB) mirrors which can strongly focus the beam down to a few micrometers. As a key parameter for many experiments, understanding of the focus characteristics and variations is demanded by users. The current instrumentation at the beamline FL24 has foreseen a dedicated Hartmann-Wavefront Sensor (HWS) to run the online, highly focused, single-shot beam characterization within the operating wavelength range of the FLASH2 [2]. However, a critical issue linked to the success of the current HWS is the assumption of high transverse coherence of the radiation. We observed a slight difference between the retrieved focuses by the HWS and those measured with the imprints method. We attribute the observed difference to the low-degree of the transverse coherence. Recently, we performed a non-destructive Young’s double-pinhole experiment, at the beamline FL24, which proved the variation of the degree of transverse coherence (25-50% deviation from the full coherence) correlated to the various machine parameters [3]. Advances in the Fresnel Diffractive Imaging (FDI) have promoted the FEL pulse characterization by reconstructing partially coherent wave fields. This approach was successfully applied to characterize highly transverse coherent, focused pulses at the beamline BL2 at the FLASH1 line [4]. We have extended the application of the FDI method, at the beamline FL24, to characterize the transverse partially coherent pulses, in a single-shot basis, and estimate a measure of the degree of the transverse coherence. Summarily, we report on the results of our previous pulse and transverse coherence characterization experiments, and discuss the feasibility of each method as an on-line photon diagnostic. Furthermore, our future plan to apply the partially coherent ptychography method [5] for the wave field characterization will be discussed providing the results of start-to-end simulations.---------------------------------------- References and links: 1. B. Faatz, et al. “The FLASH Facility: Advanced Options for FLASH2 and Future Perspectives," Applied Science 7, (2017). 2. B. Keitel, et al. “Hartmann wavefront sensors and their application at FLASH," Special Issue (PhotonDiag2015), J. Synchrotron Rad. 23, (2016). 3. T. Wodzinski, et al. “Coherence measurements with double pinholes at FLASH2," PhotonDiag 2018, Hamburg, Germany. 4. M. Mehrjoo, et al, “Single-Shot Determination of Focused FEL Wave Fields using Iterative Phase Retrieval," Opt. Express,25, (2017). 5. N. Burdet. et al, “Evaluation of partial coherence correction in X-ray ptychography ," Opt. Express, 5, (2015).

Optics letters 44(7), 1650 - 1653 (2019). doi:10.1364/OL.44.001650, Intense, ultrashort, and high-repetition-rate X-ray pulses, combined with a femtosecond optical laser, allow pump-probe experiments with fast data acquisition and femtosecond time resolution. However, the relative timing of the X-ray pulses and the optical laser pulses can be controlled only to a level of the intrinsic error of the instrument which, without characterization, limits the time resolution of experiments. This limitation inevitably calls for a precise determination of the relative arrival time, which can be used after measurement for sorting and tagging the experimental data to a much finer resolution than it can be controlled to. The observed root-mean-square timing jitter between the X-ray and the optical laser at the SPB/SFX instrument at European XFEL was 308 fs. This first measurement of timing jitter at the European XFEL provides an important step in realizing ultrafast experiments at this novel X-ray source. A method for determining the change in the complex refractive index of samples is also presented., Published by OSA, Washington, DC

Journal of synchrotron radiation 26(3), 660 - 676 (2019). doi:10.1107/S1600577519003308, The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation – in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described., Published by Wiley-Blackwell, [S.l.]

The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest.Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the studyof such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as theabsence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in alimited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information,most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied inthree dimensions, CDI can thus recover the sample’s electron density distribution. As the extension to three dimensions isaccompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservationof a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization.Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherentimaging. We present an experimental demonstration of the combination of three-dimensional structure determination throughPCDI with a cryogenically cooled biological sample—a budding yeast cell (Saccharomyces cerevisiae)—using hard (7.9 keV)synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitativethree-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and pavesthe way to future studies of unique, nonreproducible biological cells at higher resolution.

Determining fluctuations in focus properties is essential for many experiments at Self-Amplified-Spontaneous-Emission (SASE) based Free-Electron-Lasers (FELs), in particular for imaging single non-crystalline biological particles. We report on a diffractive imaging technique to fully characterize highly focused, single-shot pulses using an iterative phase retrieval algorithm, and benchmark it against an existing Hartmann wavefront sensor. The results, both theoretical and experimental, demonstrate the effectiveness of this technique to provide a comprehensive and convenient shot-to-shot measurement of focused-pulse wave fields and source-point positional variations without the need for manipulative optics between the focus and the detector.

The advantages of a novel wide dynamic range hard X-ray detector are demonstrated for (ptychographic) coherent X-ray diffractive imaging., Coherent (X-ray) diffractive imaging (CDI) is an increasingly popular form of X-ray microscopy, mainly due to its potential to produce high-resolution images and the lack of an objective lens between the sample and its corresponding imaging detector. One challenge, however, is that very high dynamic range diffraction data must be collected to produce both quantitative and high-resolution images. In this work, hard X-ray ptychographic coherent diffractive imaging has been performed at the P10 beamline of the PETRA III synchrotron to demonstrate the potential of a very wide dynamic range imaging X-ray detector (the Mixed-Mode Pixel Array Detector, or MM-PAD). The detector is capable of single photon detection, detecting fluxes exceeding 1 × 108 8-keV photons pixel−1 s−1, and framing at 1 kHz. A ptychographic reconstruction was performed using a peak focal intensity on the order of 1 × 1010 photons µm−2 s−1 within an area of approximately 325 nm × 603 nm. This was done without need of a beam stop and with a very modest attenuation, while ‘still’ images of the empty beam far-field intensity were recorded without any attenuation. The treatment of the detector frames and CDI methodology for reconstruction of non-sensitive detector regions, partially also extending the active detector area, are described.

X-ray ptychography is a rapidly developing phase retrieval technique that combines the experimental advantages of coherent diffractive imaging with the possibility to image extended specimens. Data collection requires imaging at several scan points with high positional accuracy, which implies susceptibility to mechanical drift. This is a well-known problem in ptychographic scans, which can reduce reconstruction quality and limit the achievable resolution. Using a simple model for positional drift, we show that a set of corrected positions can be found systematically, leading to strong improvements in the reconstruction of a Siemens star dataset severely affected by drift.

Optimized X-ray waveguides have been fabricated and characterized in terms of transmission, angular acceptance, farfield pattern and imaging applications. Beam confinement down to sub-10 nm in two orthogonal directions has been demonstrated, at the nano-focus endstation at P10 of PETRA III at HASYLAB/DESY., The propagation of hard X-ray synchrotron beams in waveguides with guiding layer diameters in the 9–35 nm thickness range has been studied. The planar waveguide structures consist of an optimized two-component cladding. The presented fabrication method is suitable for short and leak-proof waveguide slices with lengths (along the optical axis) in the sub-500 µm range, adapted for optimized transmission at photon energies of 11.5–18 keV. A detailed comparison between finite-difference simulations of waveguide optics and the experimental results is presented, concerning transmission, divergence of the waveguide exit beam, as well as the angular acceptance. In a second step, two crossed waveguides have been used to create a quasi-point source for propagation-based X-ray imaging at the new nano-focus endstation of the P10 coherence beamline at Petra III. By inverting the measured Fraunhofer diffraction pattern by an iterative error-reduction algorithm, a two-dimensional focus of 10 nm × 10 nm is obtained. Finally, holographic imaging of a lithographic test structure based on this optical system is demonstrated.

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·10 5 Gy, and were shown to scale with photon fluence.

Charged surfaces and ion–water interactions at an interface play a decisive role in many physico-chemical and biological processes. The classical treatment of ions at charged interfaces is the Poisson–Boltzmann (PB) theory. Despite severe simplifying assumptions it describes surprisingly well univalent ions not too close to the interface for low electrolyte concentrations in the mmol regime. However, it breaks down in the vicinity of the interface at higher surface charge densities. Consequently the list of decorations and modifications of the original PB equation is long aiming for a more realistic picture. One striking deficiency of the treatment on the pure electrostatic level is the prediction that ions of the same valence produce the same results, independent of their chemical nature. In contrast, experiments reveal pronounced differences between different ions. Specific ion effects can be found everywhere in chemistry and biology and there are many reports of pronounced differences in the properties of charged monolayers, micelles, vesicles, dispersions or polyelectrolyte multilayers using different identically charged counterions. The so-called “counterion effect” is usually discussed in terms of the Hofmeister series for cations or anions which are the result of a subtle balance of several competing evenly matched interactions. The complex interplay of electrostatics, dispersion forces, thermal motion, polarization, fluctuations, hydration, ion size effects and the impact of interfacial water structure makes it hard to identify a universal law. The diversity of specific ion effects is a direct consequence of this subtle interplay of forces and imposes a true challenge for the theories. The decisive information for an assessment of the theories is knowledge of the prevailing ion distribution. Hence a considerable amount of work has been applied to develop suitable model systems and to push surface characterization tools such as (resonant) X-ray reflectivity, total reflection X-ray fluorescence or X-ray standing waves to new limits. These techniques give direct information on the ions and on the interfacial architecture. A second alternative to complement these studies is infrared–visible sum frequency spectroscopy allowing to record surface vibrational spectra of the water as it is perturbed in the presence of the salts. The paper is organized in sections describing various facets of ion specific effects discussed within the network.

Journal of synchrotron radiation 20, 490-497 (2013). doi:10.1107/S0909049513005372, In the past decade Kirkpatrick-Baez (KB) mirrors have been established as powerful focusing systems in hard X-ray microscopy applications. Here a ptychographic characterization of the KB focus in the dedicated nano-imaging setup GINIX (Göttingen Instrument for Nano-Imaging with X-rays) at the P10 coherence beamline of the PETRA III synchrotron at HASLYLAB/DESY, Germany, is reported. More specifically, it is shown how aberrations in the KB beam, caused by imperfections in the height profile of the focusing mirrors, can be eliminated using a pinhole as a spatial filter near the focal plane. A combination of different pinhole sizes and illumination conditions of the KB setup makes the prepared optical setup well suited not only for high-resolution ptychographic coherent X-ray diffractive imaging but also for moderate-resolution/large-field-of-view propagation imaging in the divergent KB beam., Published by IUCr, Chester

Ptychographic coherent X-ray diffractive imaging (PCDI) has been combined with nano-focus X-ray diffraction to study the structure and density distribution of unstained and unsliced bacterial cells, using a hard X-ray beam of 6.2keV photon energy, focused to about 90nm by a Fresnel zone plate lens. While PCDI provides images of the bacteria with quantitative contrast in real space with a resolution well below the beam size at the sample, spatially resolved small angle X-ray scattering using the same Fresnel zone plate (cellular nano-diffraction) provides structural information at highest resolution in reciprocal space up to 2nm−1. We show how the real and reciprocal space approach can be used synergistically on the same sample and with the same setup. In addition, we present 3D hard X-ray imaging of unstained bacterial cells by a combination of ptychography and tomography. peerReviewed

We have studied the spatial coherence properties of a nano-focused x-ray beam by grating (Talbot) interferometry in projection geometry. The beam is focused by a fixed curvature mirror system optimized for high flux density under conditions of partial coherence. The spatial coherence of the divergent exit wave emitted from the mirror focus is measured by Talbot interferometry The results are compared to numerical calculations of coherence propagation. In view of imaging applications, the magnified in-line image of a test pattern formed under conditions of partial coherence is analyzed quantitatively. Finally, additional coherence filtering by use of x-ray waveguides is demonstrated. By insertion of x-ray waveguides, the beam diameter can be reduced from typical values of 200 nm to values below 15 nm. In proportion to the reduction in the focal spot size, the numerical aperture (NA) of the projection imaging system is increased, as well as the coherence length, as quantified by grating interferometry. peerReviewed

The unique strengths of x-ray microscopy are high penetration depth and near-edge resonances that provide chemical information. We use ptychography, a coherent diffractive imaging technique that disposes of the requirement for isolated specimens, and demonstrate resonant imaging by exploiting resonances near the oxygen K edge to differentiate between two oxygen-containing materials. To highlight a biological system where resonant ptychography might be used for chemical mapping of unsliced cells, reconstructions of freeze-dried Deinococcus radiodurans cells at an energy of 517 eV are shown.

Coherent x-ray diffractive microscopy enables full reconstruction of the complex transmission function of an isolated object to diffraction-limited resolution without relying on any optical elements between the sample and detector. In combination with ptychography, also specimens of unlimited lateral extension can be imaged. Here we report on an application of ptychographic coherent diffractive imaging (PCDI) in the soft x-ray regime, more precisely in the so-called water window of photon energies where the high scattering contrast between carbon and oxygen is well-suited to image biological samples. In particular, we have reconstructed the complex sample transmission function of a fossil diatom at a photon energy of 517 eV. In imaging a lithographically fabricated test sample a resolution on the order of 50 nm (half-period length) has been achieved. Along with this proof-of-principle for PCDI at soft x-ray wavelengths, we discuss the experimental and technical challenges which can occur especially for soft x-ray PCDI. peerReviewed

We have used x-ray waveguides as highly confining optical elements for nanoscale imaging of unstained biological cells using the simple geometry of in-line holography. The well-known twin-image problem is effectively circumvented by a simple and fast iterative reconstruction. The algorithm which combines elements of the classical Gerchberg-Saxton scheme and the hybrid-input-output algorithm is optimized for phase-contrast samples, well-justified for imaging of cells at multi-keV photon energies. The experimental scheme allows for a quantitative phase reconstruction from a single holographic image without detailed knowledge of the complex illumination function incident on the sample, as demonstrated for freeze-dried cells of the eukaryotic amoeba Dictyostelium discoideum. The accessible resolution range is explored by simulations, indicating that resolutions on the order of 20 nm are within reach applying illumination times on the order of minutes at present synchrotron sources.

The use of coherent x rays for microscopic imaging has seen a rapid and ongoing development within the past decade, driven by an increasing availability of highly brilliant and coherent sources worldwide. Accordingly, novel methods have been developed, which replace the microscope‘s objective lens by a numerical reconstruction scheme. The aim of the present work is to study how very recent experimental and algorithmic developments in the field can be implemented towards a highly sensitive and fully quantitative microscopy method for imaging of biological cells. To this end, different experimental approaches are studied, based on coherent far-field as well as near-field diffraction. At first, an application of the novel ptychographic imaging method to single biological cells is presented. In particular, it is demonstrated how weakly scattering biological specimens can be imaged with fully quantitative density contrast. Alongside, a sueccessful extension of the method towards soft x-ray energies is described. In the second part of the work it is shown how x-ray waveguides can be used as a point source for propagation-based microscopy of single cells in the hard x-ray regime. The specifically devised iterative reconstruction scheme allows for full quantitativity and high sensitivity and thus enables an application to single biological cells. The work contains a thorough introduction into the x-ray optical methods applied and aims at a useful and self-contained overview on aspects of signal and Fourier theory relevant for the used numerical propagation schemes. peerReviewed

We have combined two high transmission planar x-ray waveguides glued onto each other in a crossed geometry to form an effective quasi-point source. From measurements of the far-field diffraction pattern, the phase and amplitude of the near-field distribution is retrieved using the error-reduction algorithm. In agreement with finite difference field simulations (forward calculation), the reconstructed exit wave intensity distribution (inverse calculation) exhibits a full width at half maximum (FWHM) below 15 nm in both dimensions. Finally, holographic imaging is successfully demonstrated for the crossed waveguide device by translation of a lithographic test structure through the waveguide beam. peerReviewed

We report a projection phase contrast microscopy experiment using hard x-ray pink beam undulator radiation focused by an adaptive mirror system to 100--200 nm spot size. This source is used to illuminate a lithographic test pattern with a well-controlled range of spatial frequencies. The oscillatory nature of the contrast transfer function with source-to-sample distance in this holographic imaging scheme is quantified and the validity of the weak phase object approximation is confirmed for the experimental conditions.

Lensless imaging is a high potential and currently intensely targeted research goal, in view of those fields of applications for which aberration-free high-resolution lenses are not available, for example for x-ray imaging. A recently proposed (direct inversion) variant of lensless imaging combines the advantages of two classical routes toward lensless imaging, the high-resolution characteristics of iterative object reconstruction, and the direct and deterministic nature of holographic reconstruction. Here, we use a simple standard optical setup using visible wavelength, a lithographic test object and a phase-shifting reference object to demonstrate the approach. Importantly, we show that a phase-shifting reference object, instead of the absorption mask proposed earlier, is sufficient for object reconstruction. This is relevant in view of the much easier implementation in future x-ray applications. peerReviewed

Analysis of x-ray and neutron reflectivity is usually performed by modeling the density profile of the sample and performing a least square fit to the measured (phaseless) reflectivity data. Here we address the uniqueness of the reflectivity problem as well as its numerical reconstruction. In particular, we derive conditions for uniqueness, which are applicable in the kinematic limit (Born approximation), and for the most relevant case of box model profiles with Gaussian roughness. At the same time we present an iterative method to reconstruct the profile based on regularization methods. The method is successfully implemented and tested both on simulated and real experimental data.

We report an x-ray reflectivity study of phospholipid membranes deposited on silicon by vesicle fusion. The samples investigated were composed of single phospholipid bilayers as well as two-component lipid bilayer systems with varied charge density. We show that the resolution obtained in the density profile across the bilayer is high enough to distinguish two head-group maxima in the profile if the sample is in the phase coexistence regime. The water layer between the bilayer and silicon is found to depend on the lipid surface charge density.

The European X-ray Free-Electron Laser (XFEL.EU) [1] will provide ultra-short, highly-intense, coherent x-ray pulses at an unprecedented repetition rate, transforming experiments in many scientific areas, including serial femtosecond crystallography (SFX). For the purpose of SFX experiments at the XFEL.EU, a dedicated endstation is being developed to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument [2]. The setup will refocus the beam spent by SPB into a second interaction region, thereby enabling two parallel experiments. In order to overcome various challenges in XFEL crystallography, and to optimize the output for SFX experiments at XFEL.EU, the Adaptive Gain Integrating Pixel Detector (AGIPD) [3] is currently under development and is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX apparatus. The AGIPD is a hybrid-pixel detector with pixels of 200 x 200 micron^2 each. The gain of each single pixel dynamically and independently adapts to the incoming signal. Thus, diffraction patterns of high dynamic range can be recorded, with the measured signal within a single data frame ranging from single photons and up to 1e+4 photons at 12 keV. Moreover, the AGIPD is designed to store over 350 data frames from successive pulses prior to digitization and read-out, thereby enabling operation at the European XFEL with its challenging repetition rate with 10 Hz pulse trains and a 4.5 MHz intra-train repetition rate. Furthermore, the incorporation of a veto system in AGIPD will allow one to potentially store only the frames that contain diffraction data from actual crystal hits, which ultimately increases the efficiency of the detector and DAQ systems dramatically. In the present work, we will review the design of the 4Mpix AGIPD for the SFX apparatus and discuss simulations and tests of its expected performance under the conditions foreseen for SFX experiments at the XFEL.EU.

We have used a combined optical system of a high gain elliptic Kirkpatrick-Baez mirror system (KB) and a multilayer Laue lens (MLL) positioned in the focal plane of the KB for hard x-rays nano-focusing. The two-step focusing scheme is based on a high acceptance and high gain elliptical mirror with moderate focal length and a MLL with ultra-short focal length. Importantly, fabrication constraints, i.e. in mirror polishing and bending, as well as MLL deposition can be significantly relaxed, since (a) the mirror focus in the range of 200-500 nm is sufficient, and (b) the number of layers of the MLL can be correspondingly small. First demonstrations of this setup at the coherence beamline of the PETRA III storage ring yield a highly divergent far-field diffraction pattern, from which the autocorrelation function of the near-field intensity distribution was obtained. The results show that the approach is well suited to reach smallest spot sizes in the sub-10nm range at high flux. peerReviewed

We report on lensless nanoscale imaging using x-ray waveguides as ultra-small sources for quasi-point-like illumination. We first give a brief account of the basic optical setup, an overview of the progress in waveguide fabrication and characterization, as well as the basics of image formation. We then compare one-step holographic and iterative ptychographic reconstruction, both for simulated and experimental data collected on samples illuminated by waveguided beams. We demonstrate that scanning the sample with partial overlap can substantially improve reconstruction quality in holographic imaging, and that divergent beams make efficient use of the limited dynamic range of current detectors, regardless of the reconstruction scheme. Among different experimental settings presented, smallest source dimensions of 29 nm (horizontal) ×17 nm have been achieved, using multi-modal interference effects. These values have been determined by ptychographic reconstruction of a Ta test structure at 17.5 keV and have been corroborated by simulations of field propagation inside the waveguide. peerReviewed

We have carried out a ptychographic scanning coherent diffraction imaging experiment on a test object in order to characterize the hard x-ray nanobeam in a scanning x-ray microscope. In addition to a high resolution image of the test object, a detailed quantitative picture of the complex wave field in the nanofocus is obtained with high spatial resolution and dynamic range. Both are the result of high statistics due to the large number of diffraction patterns. The method yields a complete description of the focus, is robust against inaccuracies in sample positioning, and requires no particular shape or prior knowledge of the test object.

The preparation and a novel sample environment for X-ray based imaging of freeze-dried Dictyostelium discoideum cells are presented. As a first application a fluorescence imaging experiment with a nanofocused hard X-ray beam has been performed. The successful preparation was verified in elemental mappings with sub-200nm resolution, which allowed for the isolation of several ionic components specific to the cell body.

We study the bromide counterion distribution near a solid-supported monolayer in the case of vanishing bulk electrolyte concentration by resonant X-ray reflectivity. The surface charge density of the monolayer was varied by using different molar ratios of the cationic Di-Octadecyl-Di-methyl-Ammonium-Bromide (DODAB) and the neutral Di-Palmitoyl-Glycero-Phosphocholine (DPPC). The analysis, either based on a conventional box model with an additional counterion contribution, or based on an independent unbiased global optimization approach, yields a good agreement with the classical Poisson-Boltzmann theory for the salt-free case.