X-ray phase-contrast tomography of cells manipulated with an optical stretcher.

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.

Jetting bubbles observed by x-ray holography at a free-electron laser: internal structure and the effect of non-axisymmetric boundary conditions.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s00348-023-03759-9.

Micropipette aspiration as a tool for single-particle X-ray imaging and diffraction.

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.

Combined optical fluorescence microscopy and X-ray tomography reveals substructures in cell nuclei in 3D.

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.

Coherent Diffractive Imaging with Diffractive Optics.

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.

Off-axis multilayer zone plate with 16†nm × 28†nm focus for high-resolution X-ray beam induced current imaging.

Using multilayer zone plates (MZPs) as two-dimensional optics, focal spot sizes of less than 10 nm can be achieved, as we show here with a focus of 8.4 nm × 9.6 nm, but the need for order-sorting apertures prohibits practical working distances. To overcome this issue, here an off-axis illumination of a circular MZP is introduced to trade off between working distance and focal spot size. By this, the working distance between order-sorting aperture and sample can be more than doubled. Exploiting a 2D focus of 16 nm × 28 nm, real-space 2D mapping of local electric fields and charge carrier recombination using X-ray beam induced current in a single InP nanowire is demonstrated. Simulations show that a dedicated off-axis MZP can reach sub-10 nm focusing combined with reasonable working distances and low background, which could be used for in operando imaging of composition, carrier collection and strain in nanostructured devices.

Nanosecond timing and synchronization scheme for holographic pump-probe studies at the MID instrument at European XFEL.

Single-pulse holographic imaging at XFEL sources with 1012 photons delivered in pulses shorter than 100 fs reveal new quantitative insights into fast phenomena. Here, a timing and synchronization scheme for stroboscopic imaging and quantitative analysis of fast phenomena on time scales (sub-ns) and length-scales (≲100 nm) inaccessible by visible light is reported. A fully electronic delay-and-trigger system has been implemented at the MID station at the European XFEL, and applied to the study of emerging laser-driven cavitation bubbles in water. Synchronization and timing precision have been characterized to be better than 1 ns.

Combined scanning small-angle X-ray scattering and holography probes multiple length scales in cell nuclei.

X-rays are emerging as a complementary probe to visible-light photons and electrons for imaging biological cells. By exploiting their small wavelength and high penetration depth, it is possible to image whole, intact cells and resolve subcellular structures at nanometer resolution. A variety of X-ray methods for cell imaging have been devised for probing different properties of biological matter, opening up various opportunities for fully exploiting different views of the same sample. Here, a combined approach is employed to study cell nuclei of NIH-3T3 fibroblasts. Scanning small-angle X-ray scattering is combined with X-ray holography to quantify length scales, aggregation state, and projected electron and mass densities of the nuclear material. Only by joining all this information is it possible to spatially localize nucleoli, heterochromatin and euchromatin, and physically characterize them. It is thus shown that for complex biological systems, like the cell nucleus, combined imaging approaches are highly valuable.

Single-pulse phase-contrast imaging at free-electron lasers in the hard X-ray regime.

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.

3D virtual histology of human pancreatic tissue by multiscale phase-contrast X-ray tomography.

A multiscale three-dimensional (3D) virtual histology approach is presented, based on two configurations of propagation phase-contrast X-ray tomography, which have been implemented in close proximity at the GINIX endstation at the beamline P10/PETRA III (DESY, Hamburg, Germany). This enables the 3D reconstruction of characteristic morphological features of human pancreatic normal and tumor tissue, as obtained from cancer surgery, first in the form of a large-scale overview by parallel-beam illumination, followed by a zoom into a region-of-interest based on zoom tomography using a Kirkpatrick-Baez mirror with additional waveguide optics. To this end 1 mm punch biopsies of the tissue were taken. In the parallel tomography, a volumetric throughput on the order of 0.01 mm3 s-1 was achieved, while maintaining the ability to segment isolated cells. With a continuous rotation during the scan, a total acquisition time of less than 2 min was required for a full tomographic scan. Using the combination of both setups, islets of Langerhans, a three-dimensional cluster of cells in the endocrine part of the pancreas, could be located. Cells in such an islet were segmented and visualized in 3D. Further, morphological alterations of tumorous tissue of the pancreas were characterized. To this end, the anisotropy parameter Ω, based on intensity gradients, was used in order to quantify the presence of collagen fibers within the entire biopsy specimen. This proof-of-concept experiment of the multiscale approach on human pancreatic tissue paves the way for future 3D virtual pathology.

3D virtual pathohistology of lung tissue from Covid-19 patients based on phase contrast X-ray tomography.

We present a three-dimensional (3D) approach for virtual histology and histopathology based on multi-scale phase contrast x-ray tomography, and use this to investigate the parenchymal architecture of unstained lung tissue from patients who succumbed to Covid-19. Based on this first proof-of-concept study, we propose multi-scale phase contrast x-ray tomography as a tool to unravel the pathophysiology of Covid-19, extending conventional histology by a third dimension and allowing for full quantification of tissue remodeling. By combining parallel and cone beam geometry, autopsy samples with a maximum cross section of 8 mm are scanned and reconstructed at a resolution and image quality, which allows for the segmentation of individual cells. Using the zoom capability of the cone beam geometry, regions-of-interest are reconstructed with a minimum voxel size of 167 nm. We exemplify the capability of this approach by 3D visualization of diffuse alveolar damage (DAD) with its prominent hyaline membrane formation, by mapping the 3D distribution and density of lymphocytes infiltrating the tissue, and by providing histograms of characteristic distances from tissue interior to the closest air compartment.

A beamline-compatible STED microscope for combined visible-light and X-ray studies of biological matter.

A dedicated stimulated emission depletion (STED) microscope had been designed and implemented into the Göttingen Instrument for Nano-Imaging with X-rays (GINIX) at the synchrotron beamline P10 of the PETRA III storage ring (DESY, Hamburg). The microscope was installed on the same optical table used for X-ray holography and scanning small-angle X-ray scattering (SAXS). Scanning SAXS was implemented with the Kirkpatrick-Baez (KB) nano-focusing optics of GINIX, while X-ray holography used a combined KB and X-ray waveguide optical system for full-field projection recordings at a defocus position of the object. The STED optical axis was aligned (anti-)parallel to the focused synchrotron beam and was laterally displaced from the KB focus. This close proximity between the STED and the X-ray probe enabled in situ combined recordings on the same biological cell, tissue or any other biomolecular sample, using the same environment and mounting. Here, the instrumentation and experimental details of this correlative microscopy approach are described, as first published in our preceding work [Bernhardt et al. (2018), Nat. Commun. 9, 3641], and the capabilities of correlative STED microscopy, X-ray holography and scanning SAXS are illustrated by presenting additional datasets on cardiac tissue cells with labeled actin cytoskeleton.

Focus characterization of the NanoMAX Kirkpatrick-Baez mirror system.

The focusing and coherence properties of the NanoMAX Kirkpatrick-Baez mirror system at the fourth-generation MAX IV synchrotron in Lund have been characterized. The direct measurement of nano-focused X-ray beams is possible by scanning of an X-ray waveguide, serving basically as an ultra-thin slit. In quasi-coherent operation, beam sizes of down to 56 nm (FWHM, horizontal direction) can be achieved. Comparing measured Airy-like fringe patterns with simulations, the degree of coherence |µ| has been quantified as a function of the secondary source aperture (SSA); the coherence is larger than 50% for SSA sizes below 11 µm at hard X-ray energies of 14 keV. For an SSA size of 5 µm, the degree of coherence has been determined to be 87%.

Bending and Twisting Lattice Tilt in Strained Core-Shell Nanowires Revealed by Nanofocused X-ray Diffraction.

We have investigated strained GaAs-GaInP core-shell nanowires using transmission electron microscopy and nanofocused scanning X-ray diffraction. Nominally identical growth conditions for each sample were achieved by using nanoimprint lithography to create wafer-scale arrays of Au seed particles. However, we observe large individual differences, with neighboring nanowires showing either straight, bent, or twisted morphology. Using scanning X-ray diffraction, we reconstructed and quantified the bending and twisting of the nanowires in three dimensions. In one nanowire, we find that the shell lattice is tilted with respect to the core lattice, with an angle that increases from 2° at the base to 5° at the top. Furthermore, the azimuthal orientation of the tilt changes by 30° along the nanowire axis. Our results demonstrate how strained core-shell nanowire growth can lead to a rich interplay of composition, lattice mismatch, bending and lattice tilt, with additional degrees of complexity compared with thin films.

Probe reconstruction for holographic X-ray imaging.

In X-ray holographic near-field imaging the resolution and image quality depend sensitively on the beam. Artifacts are often encountered due to the strong focusing required to reach high resolution. Here, two schemes for reconstructing the complex-valued and extended wavefront of X-ray nano-probes, primarily in the planes relevant for imaging (i.e. focus, sample and detection plane), are presented and compared. Firstly, near-field ptychography is used, based on scanning a test pattern laterally as well as longitudinally along the optical axis. Secondly, any test pattern is dispensed of and the wavefront reconstructed only from data recorded for different longitudinal translations of the detector. For this purpose, an optimized multi-plane projection algorithm is presented, which can cope with the numerically very challenging setting of a divergent wavefront emanating from a hard X-ray nanoprobe. The results of both schemes are in very good agreement. The probe retrieval can be used as a tool for optics alignment, in particular at X-ray nanoprobe beamlines. Combining probe retrieval and object reconstruction is also shown to improve the image quality of holographic near-field imaging.

X-Ray Micro- and Nanodiffraction Imaging on Human Mesenchymal Stem Cells and Differentiated Cells.

Adult human mesenchymal stem cells show structural rearrangements of their cytoskeletal network during mechanically induced differentiation toward various cell types. In particular, the alignment of acto-myosin fibers is cell fate-dependent and can serve as an early morphological marker of differentiation. Quantification of such nanostructures on a mesoscopic scale requires high-resolution imaging techniques. Here, we use small- angle x-ray scattering with a spot size in the micro- and submicrometer range as a high-resolution and label-free imaging technique to reveal structural details of stem cells and differentiated cell types. We include principal component analysis into an automated empirical analysis scheme that allows the local characterization of oriented structures. Results on freeze-dried samples lead to quantitative structural information for all cell lines tested: differentiated cells reveal pronounced structural orientation and a relatively intense overall diffraction signal, whereas naive human mesenchymal stem cells lack these features. Our data support the hypothesis of stem cells establishing ordered structures along their differentiation process.

In Operando X-Ray Nanodiffraction Reveals Electrically Induced Bending and Lattice Contraction in a Single Nanowire Device.

Hard X-ray diffraction (XRD) using a nanofocused beam is used to measure both lattice contraction and bending in a single nanowire device under electric bias. The shape of the nanowire is reconstructed in 3D with sub-nanometer precision. As the bias voltage is gradually increased, nonreversible structural changes in the contact regions are observed, correlated with degradation of the electrical conductance.

Simultaneous high-resolution scanning Bragg contrast and ptychographic imaging of a single solar cell nanowire.

Simultaneous scanning Bragg contrast and small-angle ptychographic imaging of a single solar cell nanowire are demonstrated, using a nanofocused hard X-ray beam and two detectors. The 2.5 µm-long nanowire consists of a single-crystal InP core of 190 nm diameter, coated with amorphous SiO2 and polycrystalline indium tin oxide. The nanowire was selected and aligned in real space using the small-angle scattering of the 140 × 210 nm X-ray beam. The orientation of the nanowire, as observed in small-angle scattering, was used to find the correct rotation for the Bragg condition. After alignment in real space and rotation, high-resolution (50 nm step) raster scans were performed to simultaneously measure the distribution of small-angle scattering and Bragg diffraction in the nanowire. Ptychographic reconstruction of the coherent small-angle scattering was used to achieve sub-beam spatial resolution. The small-angle scattering images, which are sensitive to the shape and the electron density of all parts of the nanowire, showed a homogeneous profile along the nanowire axis except at the thicker head region. In contrast, the scanning Bragg diffraction microscopy, which probes only the single-crystal InP core, revealed bending and crystalline inhomogeneity. Both systematic and non-systematic real-space movement of the nanowire were observed as it was rotated, which would have been difficult to reveal only from the Bragg scattering. These results demonstrate the advantages of simultaneously collecting and analyzing the small-angle scattering in Bragg diffraction experiments.

Compound focusing mirror and X-ray waveguide optics for coherent imaging and nano-diffraction.

A compound optical system for coherent focusing and imaging at the nanoscale is reported, realised by high-gain fixed-curvature elliptical mirrors in combination with X-ray waveguide optics or different cleaning apertures. The key optical concepts are illustrated, as implemented 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, and examples for typical applications in biological imaging are given. Characteristic beam configurations with the recently achieved values are also described, meeting the different requirements of the applications, such as spot size, coherence or bandwidth. The emphasis of this work is on the different beam shaping, filtering and characterization methods.

Towards multi-order hard X-ray imaging with multilayer zone plates.

This article describes holographic imaging experiments using a hard X-ray multilayer zone plate (MZP) with an outermost zone width of 10 nm at a photon energy of 18 keV. An order-sorting aperture (OSA) is omitted and emulated during data analysis by a 'software OSA'. Scanning transmission X-ray microscopy usually carried out in the focal plane is generalized to the holographic regime. The MZP focus is characterized by a three-plane phase-retrieval algorithm to an FWHM of 10 nm.