Structure analysis of supra-molecular assemblies is of considerable interest in a wide range of research fields, but also extremely challenging. Biomolecular colloidal particles, such as biological vesicles or small unilamellar lipid vesicles, for example, measure only a few tens of nanometers, and need to be probed in hydrated and functionalized environments. Small-angle X-ray scattering (SAXS) is a well established technique offering high resolution in solution and at room temperature. However, due to the average over an extremely large ensemble, SAXS yields information only about the average structure (size and electron density profile). Many structural details are lost or screened by polydispersity, as well as by powder averaging. To overcome these limitations, we study single vesicles sequentially by coherent diffractive X-ray imaging using femtosecond X-ray free-electron laser (XFEL) pulses. For these experiments, single vesicles surrounded by a thin water layer are delivered into a nano-focused XFEL beam by an aerosol injector. Following the 'diffract-before-destroy' principle, the individual vesicles are probed on time scales where radiation damage has not yet set in. This approach leads to the measurement of thousands of diffraction patterns that can now be analyzed without limits associated with ensemble averaging.

X-ray microtomography at synchrotron sources is fundamentally limited by the high radiation dose applied to the samples, which restricts investigations to non-native tissue states and thereby compromises the biological relevance of the resulting data. The limitation stems from inefficient indirect detection schemes that require prolonged exposures. Efforts to extract additional contrast through multi-modal techniques, like modulation-based imaging, worsen the problem by requiring multiple tomographic scans. In addition, the techniques suffer from low modulator pattern visibility, which reduces measurement efficiency and sensitivity. We address both the detection efficiency and modulation visibility challenges using a setup that combines an X-ray waveguide, a structured phase modulator, and a photon-counting detector. Our approach simultaneously achieves near-theoretical limits in both visibility (95%) and quantum efficiency (98%), thereby enabling dose-efficient multi-modal microtomography at single-micrometer resolution. This advance represents an important step toward routine multi-modal microtomography of native-state biological specimens with the potential to advance biomedical research and disease diagnosis.

Here we report on recent progress in X-ray waveguide optics for full-field coherent imaging at the Göttingen Instrument for Nano-Imaging with X-rays (GINIX), installed at the P10 coherence beamline of the PETRA III storage ring at DESY, Hamburg. We describe fabrication methods including new materials and the corresponding characterization in terms of transmission, exit intensity distribution, far-field intensity distribution and coherence properties. In addition to single channels, which are currently used for holographic imaging, we include results on tapered waveguides and off-axis waveguide interferometers. We also address optimization of waveguide optics with respect to the novel super-resolution holography method presented by Soltau et al. [Optica (2021), 8, 818–823]. Finally, we discuss the further development of high-resolution nano-holography in view of the planned storage ring upgrade to PETRA IV.

X-ray absorption near-edge structure (XANES) spectroscopy is key to understanding functional materials. We present, to our knowledge, the first use of hard x-ray near-field holography (NFH) for three-dimensional (3D), spatially resolved XANES. While NFH provides lensless imaging with an adjustable field of view, it entails complex reconstruction. Our method jointly reconstructs NFH data acquired at multiple energies and enforces a low-dimensional spectral model based on the physically motivated assumption that each voxel spectrum is a linear combination of a small number of constituents. We demonstrate the approach on a mixed metal oxide catalyst particle, achieving accurate spectral reconstruction and revealing 3D chemical heterogeneity. XANES in NFH mode has the potential to enable nano-scale in-operando XANES microscopy with a large field of view.

Wetting of solid surfaces by a liquid is important for many natural and industrial processes, such as printing, painting, and coating. However, a quantitative description of the dynamic receding and advancing contact angle is still debated, in particular for aqueous solutions. One reason for our lack of quantitative understanding is the limited spatial resolution of currently used optical methods. We therefore present a new approach to access the submicroscopic region. We use X-ray phase contrast imaging to measure the dynamic receding contact angle on a moving glass fiber of 17 μm diameter. The fiber was pulled out of a liquid bath, which was filled with a mixture of glycerol and Milli-Q water. The dynamic receding contact angle decreased with increasing contact line velocity for all mixtures. In the holograms, we achieved a resolution of 50 nm/pixel with a spatial error of 450 nm. This spatial error is due to an extended surface region of the fiber and the liquid surface in the holograms. Our results demonstrate the feasibility of X-ray holography as a method to investigate dynamic contact angle phenomena and thereby open pathways to higher spatial and temporal resolution.

The phase shift of an electromagnetic wave, imprinted by its interaction with atomic scatterers, is a central quantity in optics and photonics. In particular, it encodes information about optical resonances and photon-matter interaction. While being a routine task in the optical regime, interferometric measurements of phase shifts in the x-ray frequency regime are notoriously challenging due to the short wavelengths and associated stability requirements. As a result, the methods demonstrated to date are unsuitable for nanoscopic systems. Here, we demonstrate a nanoscale interferometer, inspired by Young's double-slit experiment, to measure the dispersive phase shift due to the 14.4 keV nuclear resonance of the Mössbauer isotope ^57Fe coupled to an x-ray waveguide. From the single-photon interference patterns, we precisely extract the phase shifts in the vicinity of the nuclear resonance resolved in photon energy by using Bayesian inference. We find that the combined information from phase shift and absorbance reveals microscopic coupling parameters, which are not accessible from the intensity data alone. The demonstrated principle lays a basis for integrated x-ray interferometric sensors.

Wetting of solid surfaces by a liquid is important for many natural and industrial processes such as printing, painting and coating. However, a quantitative description of the dynamic receding and advancing contact angle is still debated, in particular for aqueous solutions. One reason for our lack of quantitative understanding is the limited spatial resolution of currently used optical methods. We therefore present a new approach to access the sub-microscopic region. We use X-ray phase contrast imaging to measure the dynamic receding contact angle on a moving glass fiber of 17 um diameter. The fiber was pulled out of a liquid bath which was filled with a mixture of glycerol and Milli-Q water. The dynamic receding contact angle decreased with increasing contact line velocity for all mixtures. In the holograms we achieved a resolution of 50 nm/pixel with a spatial error of 450 nm. This spatial error is due to an extended surface region of the fiber and the liquid surface in the holograms. Our results demonstrate the feasibility of X-ray holography as a method to investigate dynamic contact angle phenomena and thereby opening pathways to higher spatial and temporal resolution.

We investigate the ultrafast dynamics of plasma formation by optical breakdown, filamentation, and cavitation in water, using high spatiotemporal resolution offered by x-ray free-electron laser (XFEL) radiation. A femtosecond infrared laser pulse is focused in a water-filled cuvette and probed by a single femtosecond x-ray pulse, with a time delay covering nearly four orders of magnitude. By exploiting the quantitative contrast values obtained by phase retrieval, we can follow the transition from plasma to gas in terms of a continuous decrease of mass density in the cavity. At the same time, we image the emission of a cylindrical shock wave for the scenario of a single elongated breakdown filament with a high degree of symmetry. Contrarily, the regime of multiple breakdown spots deviates from cylindrical symmetry and the idealized picture expected for a Gaussian beam. Here different scenarios of cavitation and (collective) expansion dynamics as well as bubble fusion are observed. Specifically, we quantify the decrease of the expansion velocity with the number of auxiliary cavitation events due to a redistribution of the deposited laser energy. We also report events with (multi)filamentation reflecting instabilities in the initial distribution of the laser intensity upon formation of the plasma. Filaments with submicron diameter and few-micrometer spacing are observed, as well as the phenomena of filament emergence, splitting, and termination. The different regimes of heterogeneous optical breakdown and cavitation can be distinguished depending on the laser pulse energy. Altogether, the experiments demonstrate the potential of single-pulse XFEL imaging for the investigation of optical breakdown and ultrafast hydrodynamics. The future application of the imaging approach to soft matter environments, tissue, glasses, and opaque materials seems straightforward.

X-ray holo-tomography is a powerful tool for high-resolution three-dimensional imaging, but it faces inherent limitations in resolution caused by detector constraints, numerical aperture restrictions, and the empty beam division, which is a ubiquitous step of raw data correction in conventional inline holography and (phase contrast) μ CT. To address these challenges, we extend the technique of super-resolution holography (SRH), an iterative phase reconstruction scheme that achieves high resolution by leveraging a complex illumination model, compatible with low flux areas in the beam tail and far-field diffraction components. We include a realistic pixel block constraint and represent the fields with moderate upsampling in the effective parallel beam geometry. We then demonstrate the improvements gained in resolution and sampling efficiency experimentally, using X-ray waveguide illumination and a single photon counting detector. Our results highlight the benefits of combining computational development with experimental design and showcase the potential of SRH to improve resolution and contrast at holo-tomography beamlines.

Single bubble sonoluminescence (SBSL) is the phenomenon of synchronous light emission due to the violent collapse of a single spherical bubble in a liquid, driven by an ultrasonic field. During the bubble collapse, matter inside the bubble reaches extreme conditions of several gigapascals and temperatures on the order of 10000 K, leading to picosecond flashes of visible light. To this day, details regarding the energy focusing mechanism rely on simulations due to the fast dynamics of the bubble collapse and spatial scales below the optical resolution limit. In this work we present phase-contrast holographic imaging with single x-ray free-electron laser (XFEL) pulses of a SBSL cavitation bubble in water. X-rays probe the electron density structure and by that provide a uniquely new view on the bubble interior and its collapse dynamics. The involved fast time-scales are accessed by sub-100 fs XFEL pulses and a custom synchronization scheme for the bubble oscillator. We find that during the whole oscillation cycle the bubble's density profile can be well described by a simple step-like structure, with the radius R following the dynamics of the Gilmore model. The quantitatively measured internal density and width of the boundary layer exhibit a large variance. Smallest reconstructed bubble sizes reach down to R similar or equal to 0.8 mu m , and are consistent with spherical symmetry. While we here achieved a spatial resolution of a few 100 nm, the visibility of the bubble and its internal structure is limited by the total x-ray phase shift which can be scaled with experimental parameters.

In this work, we study the jetting dynamics of individual cavitation bubbles using x-ray holographic imaging and high-speed optical shadowgraphy. The bubbles are induced by a focused infrared laser pulse in water near the surface of a flat, circular glass plate, and later probed with ultrashort x-ray pulses produced by an x-ray free-electron laser (XFEL). The holographic imaging can reveal essential information of the bubble interior that would otherwise not be accessible in the optical regime due to obscuration or diffraction. The influence of asymmetric boundary conditions on the jet’s characteristics is analysed for cases where the axial symmetry is perturbed and curved liquid filaments can form inside the cavity. The x-ray images demonstrate that when oblique jets impact the rigid boundary, they produce a non-axisymmetric splash which grows from a moving stagnation point. Additionally, the images reveal the formation of complex gas/liquid structures inside the jetting bubbles that are invisible to standard optical microscopy. The experimental results are analysed with the assistance of full three-dimensional numerical simulations of the Navier–Stokes equations in their compressible formulation, which allow a deeper understanding of the distinctive features observed in the x-ray holographic images. In particular, the effects of varying the dimensionless stand-off distances measured from the initial bubble location to the surface of the solid plate and also to its nearest edge are addressed using both experiments and simulations. A relation between the jet tilting angle and the dimensionless bubble position asymmetry is derived. The present study provides new insights into bubble jetting and demonstrates the potential of x-ray holography for future investigations in this field.

X-rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X-ray phase-contrast imaging and tomography is combined with an X-ray-compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X-ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations.

A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet, positioned on a substrate that is structured by a hydrophobic and hydrophilic pattern to stabilize the droplet position. The substrate can support several droplets at a time. Evaporation is prevented by covering the droplet by a thin film of mineral oil. In this windowless fluid which minimizes background signal, single particles can be probed and manipulated by micropipettes, which can easily be inserted and steered in the droplet. Holographic X-ray imaging is shown to be well suited to observe and monitor the pipettes, as well as the droplet surface and the particles. Aspiration and force generation are also enabled based on an application of controlled pressure differences. Experimental challenges are addressed and first results are presented, obtained at two different undulator endstations with nano-focused beams. Finally, the sample environment is discussed in view of future coherent imaging and diffraction experiments with synchrotron radiation and single X-ray free-electron laser pulses.

We explore the pressure evolution and structural dynamics of transient phase transitions in a microfluidic water jet after laser-induced dielectric breakdown. To this end, we use a combined approach of near-field holography with single femtosecond x-ray free-electron laser pulses and x-ray diffraction. During cavitation and jet breakup, we observe shock wave emission along the jet. The formation of the shockwave is accompanied by pronounced changes in the structure factor of water as an evidence by a shift in the water diffraction peak. This indicates a transition to a high density liquid structure induced by the transient pressure increase.

We present a novel approach to x-ray microscopy based on a multilayer zone plate which is positioned behind a sample similar to an objective lens. However, unlike transmission x-ray microscopy, we do not content ourselves with a sharp intensity image; instead, we incorporate the multilayer zone plate transfer function directly in an iterative phase retrieval scheme to exploit the large diffraction angles of the small layers. The presence of multiple diffraction orders, which is conventionally a nuisance, now comes as an advantage for the reconstruction and photon efficiency. In a first experiment, we achieve sub-10-nm resolution and a quantitative phase contrast.

The function of a biological cell is fundamentally defined by the structural architecture of packaged DNA in the nucleus. Elucidating information about the packaged DNA is facilitated by high-resolution imaging. Here, we combine and correlate hard X-ray propagation-based phase contrast tomography and visible light confocal microscopy in three dimensions to probe DNA in whole cell nuclei of NIH-3T3 fibroblasts. In this way, unlabeled and fluorescently labeled substructures within the cell are visualized in a complementary manner. Our approach enables the quantification of the electron density, volume and optical fluorescence intensity of nuclear material. By joining all of this information, we are able to spatially localize and physically characterize both active and inactive heterochromatin, euchromatin, pericentric heterochromatin foci and nucleoli.

Huntington's disease (HD) is one of nine neurodegenerative disorders associated with an extension of polyglutamine (polyQ) in proteins. In HD, the polyQ tract in the huntingtin protein is extended beyond a threshold of 38 amino acids leading to the formation of amyloidal structures in the cytoplasm and nucleus. We investigated here the structure of Htt (Huntingtin) amyloid fibrils incellulo with nanoprobe small angle X-ray scattering. As these measurements were performed under cryogenic conditions, the information is obtained on the aggregates in their natural, hydrated environment without the need of staining and chemical fixation. We also could show the presence of oligomer structures not visible in fluorescence microscopy. Structural information on repetitive units inside of Htt inclusion bodies was determined from the SAXS data and compared to electron microscopy images. The results suggest that nanoprobe cryo-SAXS can serve as powerful tool to investigate the kinetics of amyloid aggregate formation inside cells and to understand how fibril formation can be influenced by drugs and other external stimuli.

X-ray free-electron lasers (XFELs) have opened up unprecedented opportunities for time-resolved nano-scale imaging with X-rays. Near-field propagation-based imaging, and in particular near-field holography (NFH) in its high-resolution implementation in cone-beam geometry, can offer full-field views of a specimen's dynamics captured by single XFEL pulses. To exploit this capability, for example in optical-pump/X-ray-probe imaging schemes, the stochastic nature of the self-amplified spontaneous emission pulses, i.e. the dynamics of the beam itself, presents a major challenge. In this work, a concept is presented to address the fluctuating illumination wavefronts by sampling the configuration space of SASE pulses before an actual recording, followed by a principal component analysis. This scheme is implemented at the MID (Materials Imaging and Dynamics) instrument of the European XFEL and time-resolved NFH is performed using aberration-corrected nano-focusing compound refractive lenses. Specifically, the dynamics of a micro-fluidic water-jet, which is commonly used as sample delivery system at XFELs, is imaged. The jet exhibits rich dynamics of droplet formation in the break-up regime. Moreover, pump–probe imaging is demonstrated using an infrared pulsed laser to induce cavitation and explosion of the jet.

X-ray in-line holography is well suited for three-dimensional imaging, since it covers a large field of view without the necessity of scanning. However, its resolution does not extend to the range covered by coherent diffractive imaging or ptychography. In this work, we show full-field holographic x-ray imaging based on cone-beam illumination, beyond the resolution limit given by the cone-beam numerical aperture. Image information encoded in far-field diffraction and in holographic self-interference is treated in a common reconstruction scheme, without the usual empty beam correction step of in-line holography. An illumination profile tailored by waveguide optics and exactly known by prior probe retrieval is shown to be sufficient for solving the phase problem. The approach paves the way toward high-resolution and dose-efficient x-ray tomography, well suited for the current upgrades of synchrotron radiation sources to diffraction-limited storage rings.