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Cavitation bubbles show many nonlinear characteristics of liquid and gas phase. Here the authors demonstrate coherent diffractive imaging of cavitation bubble using infrared pump and XFEL probe and discuss the formation and evolution of the bubbles.

X-ray techniques benefit from correlative imaging approaches, but combination with super-resolution microscopy has not been explored. Here the authors image the cardiomyocyte cytoskeleton by combining holographic X-ray imaging, X-ray scanning diffraction and STED in the same synchrotron endstation.

Abstract We present an approach towards four dimensional (4d) movies of materials, showing dynamic processes within the entire 3d structure. The method is based on tomographic reconstruction on dynamically curved paths using a motion model estimated by optical flow techniques, considerably reducing the typical motion artefacts of dynamic tomography. At the same time we exploit x-ray phase contrast based on free propagation to enhance the signal from micron scale structure recorded with illumination times down to a millisecond (ms). The concept is demonstrated by observing the burning process of a match stick in 4d, using high speed synchrotron phase contrast x-ray tomography recordings. The resulting movies reveal the structural changes of the wood cells during the combustion.

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.

We have carried out time resolved stroboscopic diffraction experiments on standing surface acoustic waves (SAWs) of Rayleigh type on a LiNbO3 substrate. A novel timing system has been developed and commissioned at the storage ring Petra III of Desy, allowing for phase locked stroboscopic diffraction experiments applicable to a broad range of timescales and experimental conditions. The combination of atomic structural resolution with temporal resolution on the picosecond time scale allows for the observation of the atomistic displacements for each time (or phase) point within the SAW period. A seamless transition between dynamical and kinematic scattering regimes as a function of the instantaneous surface amplitude induced by the standing SAW is observed. The interpretation and control of the experiment, in particular disentangling the diffraction effects (kinematic to dynamical diffraction regime) from possible non-linear surface effects is unambiguously enabled by the precise control of phase between the standing SAW and the synchrotron bunches. The example illustrates the great flexibility and universality of the presented timing system, opening up new opportunities for a broad range of time resolved experiments.

We have implemented a time-dependent (dynamic) x-ray tomography of sedimenting micro-spheres suspended in water. To achieve phase contrast at high magnification we use the divergent and highly coherent beam emitted from an x-ray waveguide. Holograms are recorded with 5 ms acquisition time while the sample is rotated at 1 Hz, over a run of 40 s. We show that under these conditions, more than 20 000 individual particle trajectories can be tracked. The analysis of the trajectories shows apparent super-diffusive behavior due to collective flow patterns, as also further evidenced by plotting the temporal averaged spatial distribution of particle densities and velocities.

X-ray diffraction from biomolecular assemblies is a powerful technique which can provide structural information about complex architectures such as the locomotor systems underlying muscle contraction. However, in its conventional form, macromolecular diffraction averages over large ensembles. Progress in x-ray optics has now enabled to probe structures on sub-cellular scales, with the beam confined to a distinct organelle. Here, we use scanning small angle x-ray scattering (scanning SAXS) to probe the diffraction from cytoskeleton networks in cardiac tissue cells. In particular, we focus on actin-myosin composites, which we identify as the dominating contribution to the anisotropic diffraction patterns, by correlation with optical fluorescence microscopy. To this end, we use a principal component analysis approach to quantify direction, degree of orientation, nematic order, and the second moment of the scattering distribution in each scan point. We compare the fiber orientation from micrographs of fluorescently labeled actin fibers to the structure orientation of the x-ray dataset and thus correlate signals of two different measurements: the native electron density distribution of the local probing area versus specifically labeled constituents of the sample. Further, we develop a robust and automated fitting approach based on a power law expansion, in order to describe the local structure factor in each scan point over a broad range of the momentum transfer ${q}_{{\rm{r}}}$ . Finally, we demonstrate how the methodology shown for freeze dried cells in the first part of the paper can be translated to alive cell recordings.

Image reconstruction of in-line holography depends crucially on the probing wave front used to illuminate an object. Aberrations inherent to the illumination can mix with the features imposed by the object. Conventional raw data processing methods rely on the division of the measured hologram by the intensity profile of the probe and are not able to fully eliminate artifacts caused by the illumination. Here we present a generalized ptychography approach to simultaneously reconstruct object and probe in the optical near-field. Combining the ideas of ptychographic lateral shifts of the object with variations of the propagation distance by longitudinal shifts, simultaneous reconstruction of object and probe was achieved equally well for a highly aberrated and a mildly disturbed probe without the need for an additional wave front diffuser. The method overcomes the image deterioration by a non-ideal probe and at the same time any restrictions due to linearization of the object’s transmission function or the Fresnel propagator. The method is demonstrated experimentally using visible light and hard x-rays, in both parallel beam and cone beam geometry, which is relevant for high resolution x-ray imaging. It also opens up a new approach to characterize extended wave fronts by phase retrieval.

The synaptic vesicle cluster (SVC) is an essential component of chemical synapses, which provides neurotransmitter-loaded vesicles during synaptic activity, at the same time as also controlling the local concentrations of numerous exo- and endocytosis cofactors. In addition, the SVC hosts molecules that participate in other aspects of synaptic function, from cytoskeletal components to adhesion proteins, and affects the location and function of organelles such as mitochondria and the endoplasmic reticulum. We argue here that these features extend the functional involvement of the SVC in synapse formation, signalling and plasticity, as well as synapse stabilization and metabolism. We also propose that changes in the size of the SVC coalesce with changes in the postsynaptic compartment, supporting the interplay between pre- and postsynaptic dynamics. Thereby, the SVC could be seen as an 'all-in-one' regulator of synaptic structure and function, which should be investigated in more detail, to reveal molecular mechanisms that control synaptic function and heterogeneity., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background and Aim: Full-thickness biopsies of the intestinal wall may be used to study and assess damage to the neurons of the enteric nervous system (ENS), that is, enteric neuropathy. The ENS is difficult to examine due to its localization deep in the intestinal wall and its organization with several connections in diverging directions. Histological sections used in clinical practice only visualize the sample in a two-dimensional way. X-ray phase-contrast micro-computed tomography (PC-μCT) has shown potential to assess the cross-sectional thickness and volume of the ENS in three dimensions (3D). The aim of this study was to explore the potential of PC-μCT to evaluate its use to determine the size of the ENS., Methods: Full-thickness biopsies of ileum obtained during surgery from five controls and six patients clinically diagnosed with enteric neuropathy and dysmotility were included. Punch biopsies of 1 mm in diameter and 1 cm in length, from an area containing myenteric plexus, were extracted from paraffin blocks, and scanned with synchrotron-based PC-μCT without any staining., Results: The microscopic volumetric structure of the neural tissue (consisting of both ganglia and fascicles) could be determined in all samples. The ratio of neural tissue volume/total tissue volume was higher in controls than in patients with enteric neuropathy ( P  = 0.013). The patient with the longest disease duration had the lowest ratio., Conclusion: The assessment of neural tissue can be performed in an objective, standardized way, to ensure reproducibility and comparison under physiological and pathological conditions. Further evaluation is needed to examine the role of this method in the diagnosis of enteric neuropathy., (© 2024 The Author(s). JGH Open published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Cardiac function relies on the autonomous molecular contraction mechanisms in the ventricular wall. Contraction is driven by ordered motor proteins acting in parallel to generate a macroscopic force. The averaged structure can be investigated by diffraction from model tissues such as trabecular and papillary cardiac muscle using collimated synchrotron beams, offering high resolution in reciprocal space. In the ventricular wall, however, the muscle tissue is compartmentalized into smaller branched cardiomyocytes, with a higher degree of disorder. We show that X-ray diffraction is now also capable of resolving the structural organization of actomyosin in single isolated cardiomyocytes of the ventricular wall. In addition to the hexagonal arrangement of thick and thin filaments, the diffraction signal of the hydrated and fixated cardiomyocytes was sufficient to reveal the myosin motor repeat (M3), the troponin complex repeat (Tn), and the sarcomere length. The sarcomere length signal comprised up to 13 diffraction orders, which were used to compute the sarcomere density profile based on Fourier synthesis. The Tn and M3 spacings were found in the same range as previously reported for other muscle types. The approach opens up a pathway to record the structural dynamics of living cells during the contraction cycle, toward a more complete understanding of cardiac muscle function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (open access.)

X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory μ CT radiation, more easily accessible for pre-clinical research., (© 2024. The Author(s).)

A minimal diffusion barrier is key to the pulmonary gas exchange. In alveolar capillary dysplasia (ACD), a rare genetically driven disease of early infancy, this crucial fibrovascular interface is compromised while the underlying pathophysiology is insufficiently understood. Recent in-depth analyses of vascular alterations in adult lung disease encouraged researchers to extend these studies to ACD and compare the changes of the microvasculature. Lung tissue samples of children with ACD (n = 12), adults with non-specific interstitial pneumonia (n = 12), and controls (n = 20) were studied using transmission electron microscopy, single-gene sequencing, immunostaining, exome sequencing, and broad transcriptome profiling. In ACD, pulmonary capillary basement membranes were hypertrophied, thickened, and multilamellated. Transcriptome profiling revealed increased CDH5, COL4A1, COL15A1, PTK2B, and FN1 and decreased VIT expression, confirmed by immunohistochemistry. In contrast, non-specific interstitial pneumonia samples showed a regular basement membrane architecture with preserved VIT expression but also increased COL15A1 + vessels. This study provides insight into the ultrastructure and pathophysiology of ACD. The lack of normally developed lung capillaries appeared to cause a replacement by COL15A1 + vessels, a mechanism recently described in interstitial lung disease. The VIT loss and FN1 overexpression might contribute to the unique appearance of basement membranes in ACD. Future studies are needed to explore the therapeutic potential of down-regulating the expression of FN1 and balancing VIT deficiency., Competing Interests: Disclosure Statement J.C.S. received fees for lectures from Boehringer Ingelheim and Kinevant, all outside the present study. T.W. declares funding by the German Ministry of Research and Education. M.M.H. received fees for lectures and consultations from Acceleron, Actelion, AOP, Bayer, Janssen, MSD, and Pfizer, all outside the present study. J.F. received personal fees/speaker honoraria from AstraZeneca, outside the submitted work. M.G. received fees for lectures from Boehringer Ingelheim, participates in an adjudication board in a nintedanib clinical trial, and received a research grant from Boehringer Ingelheim. D.D.J. received fees for lectures from Boehringer Ingelheim and declares a research contract with Boehringer Ingelheim (contract number 43099358). All other authors declare no existing conflicts of interest., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We study the formation of vesicle condensates induced by the protein synapsin, as a cell-free model system mimicking vesicle pool formation in the synapse. The system can be considered as an example of liquid-liquid phase separation (LLPS) in biomolecular fluids, where one phase is a complex fluid itself consisting of vesicles and a protein network. We address the pertinent question why the LLPS is self-limiting and stops at a certain size, i.e., why macroscopic phase separation is prevented. Using fluorescence light microscopy, we observe different morphologies of the condensates (aggregates) depending on the protein-to-lipid ratio. Cryogenic electron microscopy then allows us to resolve individual vesicle positions and shapes in a condensate and notably the size and geometry of adhesion zones between vesicles. We hypothesize that the membrane tension induced by already formed adhesion zones then in turn limits the capability of vesicles to bind additional vesicles, resulting in a finite condensate size. In a simple numerical toy model we show that this effect can be accounted for by redistribution of effective binding particles on the vesicle surface, accounting for the synapsin-induced adhesion zone., (© 2024. The Author(s).)

Background: Phasmatodea are well known for their ability to disguise themselves by mimicking twigs, leaves, or bark, and are therefore commonly referred to as stick and leaf insects. In addition to this and other defensive strategies, many phasmatodean species use paired prothoracic repellent glands to release defensive chemicals when disturbed by predators or parasites. These glands are considered as an autapomorphic trait of the Phasmatodea. However, detailed knowledge of the gland anatomy and chemical compounds is scarce and only a few species were studied until now. We investigated the repellent glands for a global sampling of stick and leaf insects that represents all major phasmatodean lineages morphologically via µCT scans and analyzed the anatomical traits in a phylogenetic context., Results: All twelve investigated species possess prothoracic repellent glands that we classify into four distinct gland types. 1: lobe-like glands, 2: sac-like glands without ejaculatory duct, 3: sac-like glands with ejaculatory duct and 4: tube-like glands. Lobe-like glands are exclusively present in Timema, sac-like glands without ejaculatory duct are only found in Orthomeria, whereas the other two types are distributed across all other taxa (= Neophasmatodea). The relative size differences of these glands vary significantly between species, with some glands not exceeding in length the anterior quarter of the prothorax, and other glands extending to the end of the metathorax., Conclusions: We could not detect any strong correlation between aposematic or cryptic coloration of the examined phasmatodeans and gland type or size. We hypothesize that a comparatively small gland was present in the last common ancestor of Phasmatodea and Euphasmatodea, and that the gland volume increased independently in subordinate lineages of the Occidophasmata and Oriophasmata. Alternatively, the stem species of Neophasmatodea already developed large glands that were reduced in size several times independently. In any case, our results indicate a convergent evolution of the gland types, which was probably closely linked to properties of the chemical components and different predator selection pressures. Our study is the first showing the great anatomical variability of repellent glands in stick and leaf insects., (© 2023. The Author(s).)

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., Competing Interests: Conflict of interestThe authors have no conflicts of interest to declare that are relevant to the content of this article., (© The Author(s) 2024.)

Vesicle pools can form by attractive interaction in a solution, mediated by proteins or divalent ions such as calcium. The pools, which are alternatively also denoted as vesicle clusters, form by liquid-liquid phase separation (LLPS) from an initially homogeneous solution. Due to the short range liquid-like order of vesicles in the pool or cluster, the vesicle-rich phase can also be regarded as a condensate, and one would like to better understand not only the structure of these systems, but also their dynamics. The diffusion of vesicles, in particular, is expected to change when vesicles are arrested in a pool. Here we investigate whether passive microrheology based on X-ray photon correlation spectroscopy (XPCS) is a suitable tool to study model systems of artificial lipid vesicles exhibiting LLPS, and more generally also other heterogeneous biomolecular fluids. We show that by adding highly scattering tracer particles to the solution, valuable information on the single vesicle as well as collective dynamics can be inferred. While the correlation functions reveal freely diffusing tracer particles in solutions at low CaCl[Formula: see text] concentrations, the relaxation rate [Formula: see text] shows a nonlinear dependence on [Formula: see text] at a higher concentration of around 8 mM CaCl[Formula: see text], characterised by two linear regimes with a broad cross-over. We explain this finding based on arrested diffusion in percolating vesicle clusters., (© 2023. The Author(s).)

Aims: Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity and sensitivity. Ideally, such methods should be performed within intact cardiac tissue with high relevance for humans and detect acute and chronic side effects on electrophysiological behaviour, contractility, and tissue structure in an unbiased manner. Herein, we evaluate healthy pig myocardial slices and biomimetic cultivation setups (BMCS) as a new cardiotoxicity screening approach., Methods and Results: Pig left ventricular samples were cut into slices and spanned into BMCS with continuous electrical pacing and online force recording. Automated stimulation protocols were established to determine the force-frequency relationship (FFR), frequency dependence of contraction duration, effective refractory period (ERP), and pacing threshold. Slices generated 1.3 ± 0.14 mN/mm2 force at 0.5 Hz electrical pacing and showed a positive FFR and a shortening of contraction duration with increasing pacing rates. Approximately 62% of slices were able to contract for at least 6 days while showing stable ERP, contraction duration-frequency relationship, and preserved cardiac structure confirmed by confocal imaging and X-ray diffraction analysis. We used specific blockers of the most important cardiac ion channels to determine which analysis parameters are influenced. To validate our approach, we tested five drug candidates selected from the Comprehensive in vitro Proarrhythmia Assay list as well as acetylsalicylic acid and DMSO as controls in a blinded manner in three independent laboratories. We were able to detect all arrhythmic drugs and their respective mode of action on cardiac tissue including inhibition of Na+, Ca2+, and hERG channels as well as Na+/Ca2+ exchanger., Conclusion: We systematically evaluate this approach for cardiotoxicity screening, which is of high relevance for humans and can be upscaled to medium-throughput screening. Thus, our approach will improve the predictive value and efficiency of preclinical cardiotoxicity screening., Competing Interests: Conflict of interest: A.D. and Th.S. are shareholders of InVitroSys GmbH. There are no other remaining competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Purpose: Assessing the complex three-dimensional (3D) structure of the cochlea is crucial to understanding the fundamental aspects of signal transduction in the inner ear and is a prerequisite for the development of novel cochlear implants. X-ray phase-contrast computed tomography offers destruction-free 3D imaging with little sample preparation, thus preserving the delicate structure of the cochlea. The use of heavy metal stains enables higher contrast and resolution and facilitates segmentation of the cochlea., Approach: For μ-CT of small animal and human cochlea, we explore the heavy metal osmium tetroxide (OTO) as a radiocontrast agent and delineate laboratory μ - CT from synchrotron CT. We investigate how phase retrieval can be used to improve the image quality of the reconstructions, both for stained and unstained specimens., Results: Image contrast for soft tissue in an aqueous solution is insufficient under the in-house conditions, whereas the OTO stain increases contrast for lipid-rich tissue components, such as the myelin sheaths in nervous tissue, enabling contrast-based rendering of the different components of the auditory nervous system. The overall morphology of the cochlea with the three scalae and membranes is very well represented. Further, the image quality of the reconstructions improves significantly when a phase retrieval scheme is used, which is also suitable for non-ideal laboratory μ - CT settings. With highly brilliant synchrotron radiation (SR), we achieve high contrast for unstained whole cochleae at the cellular level., Conclusions: The OTO stain is suitable for 3D imaging of small animal and human cochlea with laboratory μ - CT , and relevant pathologies, such as a loss of sensory cells and neurons, can be visualized. With SR and optimized phase retrieval, the cellular level can be reached even for unstained samples in aqueous solution, as demonstrated by the high visibility of single hair cells and spiral ganglion neurons., (© 2023 The Authors.)

Purpose: X-ray phase-contrast tomography (XPCT) is a non-destructive, three-dimensional imaging modality that provides higher contrast in soft tissue than absorption-based CT and allows one to cover the cytoarchitecture from the centi- and millimeter scale down to the nanoscale. To further increase contrast and resolution of XPCT, for example, in view of addressing connectivity issues in the central nervous system (CNS), metal staining is indispensable. However, currently used protocols, for example, based on osmium and/or uranium are less suited for XPCT, due to an excessive β / δ -ratio. In this work, we explore the suitability of different staining agents for XPCT. Particularly, neodymium(III)-acetate (NdAc), which has recently been proposed as a non-toxic, non-radioactive easy to use alternative contrast agent for uranyl acetate (UAc) in electron microscopy, is investigated. Due to its vertical proximity to UAc in the periodic table, similar chemical but better suited optical properties for phase contrast can be expected., Approach: Differently stained whole eye samples of wild type mouse and tissues of the CNS are embedded into EPON epoxy resin and scanned using synchrotron as well as with laboratory radiation. Phase retrieval is performed on the projection images, followed by tomographic reconstruction, which enables a quantitative analysis based on the reconstructed electron densities. Segmentation techniques and rendering software is used to visualize structures of interest in the sample., Results: We show that staining neuronal samples with NdAc enhances contrast, in particular for laboratory scans, allowing high-resolution imaging of biological soft tissue in-house. For the example of murine retina, specifically rods and cones as well as the sclera and the Ganglion cell layer seem to be targeted by the stain. A comparison of electron density by the evaluation of histograms allowed to determine quantitative measures to describe the difference between the examined stains., Conclusion: The results suggest NdAc to be an effective stain for XPCT, with a preferential binding to anionic groups, such as phosphate and carboxyl groups at cell surfaces, targeting certain layers of the retina with a stronger selectivity compared to other staining agents. Due to the advantageous X-ray optical properties, the stain seems particularly well-suited for phase contrast, with a comparably small number density and an overall superior image quality at laboratory sources., (© 2023 The Authors.)

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., (open access.)

We investigate structural properties of neurons in the granular layer of human cerebellum with respect to their involvement in multiple sclerosis (MS). To this end we analyze data recorded by X-ray phase contrast tomography from tissue samples collected post mortem from a MS and a healthy control group. Using automated segmentation and histogram analysis based on optimal transport theory (OT) we find that the distributions representing nuclear structure in the granular layer move to a more compact nuclear state, i.e. smaller, denser and more heterogeneous nuclei in MS. We have previously made a similar observation for neurons of the dentate gyrus in Alzheimer's disease, suggesting that more compact structure of neuronal nuclei which we attributed to increased levels of heterochromatin, may possibly represent a more general phenomenon of cellular senescence associated with neurodegeneration., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives. As the central organ of the respiratory system, the human lung is responsible for supplying oxygen to the blood, which reaches the erythrocytes by diffusion through the alveolar walls and is then distributed throughout the body. By exploiting the difference in electron density detected by a phase shift in soft tissue, high-resolution x-ray phase-contrast computed tomography (XPCT) can resolve biological structures in a sub- μ m range, shedding new light on the three-dimensional structure of the lungs, physiological functions and pathological mechanisms. Approach. This work presents both synchrotron and laboratory XPCT results of postmortem tissue from autopsies and biopsies embedded with various preparation protocols such as precision-cut lung slices, cryogenically fixed lung tissue, as well as paraffin and alcohol fixed tissue. The selection of pathological abnormalities includes channel of Lambert, bronchus-associated lymphoid tissue and alveolar capillary dysplasia with misalignment of pulmonary veins. Subsequently, quantification and visualization approaches are presented. Main results. The overall high image quality even of in-house XPCT scans for the case of FFPE biopsies can be exploited for a wide range of pulmonary pathologies and translated to dedicated and optimized instrumentation which could be operated in clinical setting. By using synchrotron radiation, contrast can be further increased to resolve sub- μ m sized features down to the sub-cellular level. The results demonstrate that a wide range of preparation protocols including sample mounting in liquids can be used. Significance. With XPCT, poorly understood 3D structures can be identified in larger volume overview and subsequently studied in more detail at higher resolution. With the full 3D structure, the respective physiological functions of airways or vascular networks, and the different pathophysiologic mechanisms can be elucidated or at least underpinned with structural data. Moreover, synchrotron data can be used to validate laboratory protocols and provide ground truth for standardizing the method., (Creative Commons Attribution license.)

Background: The worldwide increase of pancreatic ductal adenocarcinoma (PDAC), which still has one of the lowest survival rates, requires novel imaging tools to improve early detection and to refine diagnosis. Therefore, the aim of this study was to assess the feasibility of propagation-based phase-contrast X-ray computed tomography of already paraffin-embedded and unlabeled human pancreatic tumor tissue to achieve a detailed three-dimensional (3D) view of the tumor sample in its entirety., Methods: Punch biopsies of areas of particular interest were taken from paraffin blocks after initial histological analysis of hematoxylin and eosin stained tumor sections. To cover the entire 3.5 mm diameter of the punch biopsy, nine individual tomograms with overlapping regions were acquired in a synchrotron parallel beam configuration and stitched together after data reconstruction. Due to the intrinsic contrast based on electron density differences of tissue components and a voxel size of 1.3 μm achieved PDAC and its precursors were clearly identified., Results: Characteristic tissue structures for PDAC and its precursors, such as dilated pancreatic ducts, altered ductal epithelium, diffuse immune cell infiltrations, increased occurrence of tumor stroma and perineural invasion were clearly identified. Certain structures of interest were visualized in three dimensions throughout the tissue punch. Pancreatic duct ectasia of different caliber and atypical shape as well as perineural infiltration could be contiguously traced by viewing serial tomographic slices and by applying semi-automatic segmentation. Histological validation of corresponding sections confirmed the former identified PDAC features., Conclusion: In conclusion, virtual 3D histology via phase-contrast X-ray tomography visualizes diagnostically relevant tissue structures of PDAC in their entirety, preserving tissue integrity in label-free, paraffin embedded tissue biopsies. In the future, this will not only enable a more comprehensive diagnosis but also a possible identification of new 3D imaging tumor markers., (© 2023. The Author(s).)

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + -macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations., (© 2022. The Author(s).)

Improved hearing restoration by cochlear implants (CI) is expected by optical cochlear implants (oCI) exciting optogenetically modified spiral ganglion neurons (SGNs) via an optical pulse generated outside the cochlea. The pulse is guided to the SGNs inside the cochlea via flexible polymer-based waveguide probes. The fabrication of these waveguide probes is realized by using 6" wafer-level micromachining processes, including lithography processes such as spin-coating cladding layers and a waveguide layer in between and etch processes for structuring the waveguide layer. Further adhesion layers and metal layers for laser diode (LD) bonding and light-outcoupling structures are also integrated in this waveguide process flow. Optical microscope and SEM images revealed that the majority of the waveguides are sufficiently smooth to guide light with low intensity loss. By coupling light into the waveguides and detecting the outcoupled light from the waveguide, we distinguished intensity losses caused by bending the waveguide and outcoupling. The probes were used in first modules called single-beam guides (SBGs) based on a waveguide probe, a ball lens and an LD. Finally, these SBGs were tested in animal models for proof-of-concept implantation experiments.

The size, polydispersity, and electron density profile of synaptic vesicles (SVs) can be studied by small-angle X-ray scattering (SAXS), i.e. by X-ray diffraction from purified SV suspensions in solution. Here we show that size and shape transformations, as they appear in the functional context of these important synaptic organelles, can also be monitored by SAXS. In particular, we have investigated the active uptake of neurotransmitters, and find a mean vesicle radius increase of about 12% after the uptake of glutamate, which indicates an unusually large extensibility of the vesicle surface, likely to be accompanied by conformational changes of membrane proteins and rearrangements of the bilayer. Changes in the electron density profile (EDP) give first indications for such a rearrangement. Details of the protein structure are screened, however, by SVs polydispersity. To overcome the limitations of large ensemble averages and heterogeneous structures, we therefore propose serial X-ray diffraction by single free electron laser pulses. Using simulated data for realistic parameters, we show that this is in principle feasible, and that even spatial distances between vesicle proteins could be assessed by this approach., (© 2022. The Author(s).)

Based on phase retrieval, lensless coherent imaging and in particular holography offers quantitative phase and amplitude images. This is of particular importance for spectral ranges where suitable lenses are challenging, such as for hard x-rays. Here, we propose a phase retrieval approach for inline x-ray holography based on Tikhonov regularization applied to the full nonlinear forward model of image formation. The approach can be seen as a nonlinear generalization of the well-established contrast transfer function (CTF) reconstruction method. While similar methods have been proposed before, the current work achieves nonlinear, constrained phase retrieval at competitive computation times. We thus enable high-throughput imaging of optically strong objects beyond the scope of CTF. Using different examples of inline holograms obtained from illumination by a x-ray waveguide-source, we demonstrate superior image quality even for samples which do not obey the assumption of a weakly varying phase. Since the presented approach does not rely on linearization, we expect it to be well suited also for other probes such as visible light or electrons, which often exhibit strong phase interaction.

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., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Background: The enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm., Aim: To examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS., Methods: Six patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton ® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples., Results: In the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings., Conclusion: The analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

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.

For the first time, we have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross-section of 3.5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility the synchrotron in a parallel beam configuration. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high-resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in the form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis., Competing Interests: MR, PM, VA, AB, MA, HS, FL, CW, MK, DJ, TS No competing interests declared, (© 2021, Reichardt et al.)

We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer's disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

In this work, we optimize the setups and experimental parameters of X-ray phase-contrast computed-tomography for the three-dimensional imaging of the cyto- and myeloarchitecture of cerebral cortex, including both human and murine tissue. We present examples for different optical configurations using state-of-the art synchrotron instruments for holographic tomography, as well as compact laboratory setups for phase-contrast tomography in the direct contrast (edge-enhancement) regime. Apart from unstained and paraffin-embedded tissue, we tested hydrated tissue, as well as heavy metal stained and resin-embedded tissue using two different protocols. Further, we show that the image quality achieved allows to assess the neuropathology of multiple sclerosis in a biopsy sample collected during surgery., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Incoherent diffractive imaging (IDI) promises structural analysis with atomic resolution based on intensity interferometry of pulsed X-ray fluorescence emission. However, its experimental realization is still pending and a comprehensive theory of contrast formation has not been established to date. Explicit expressions are derived for the equal-pulse two-point intensity correlations, as the principal measured quantity of IDI, with full control of the prefactors, based on a simple model of stochastic fluorescence emission. The model considers the photon detection statistics, the finite temporal coherence of the individual emissions, as well as the geometry of the scattering volume. The implications are interpreted in view of the most relevant quantities, including the fluorescence lifetime, the excitation pulse, as well as the extent of the scattering volume and pixel size. Importantly, the spatiotemporal overlap between any two emissions in the sample can be identified as a crucial factor limiting the contrast and its dependency on the sample size can be derived. The paper gives rigorous estimates for the optimum sample size, the maximum photon yield and the expected signal-to-noise ratio under optimal conditions. Based on these estimates, the feasibility of IDI experiments for plausible experimental parameters is discussed. It is shown in particular that the mean number of photons per detector pixel which can be achieved with X-ray fluorescence is severely limited and as a consequence imposes restrictive constraints on possible applications., (open access.)

Cavitation bubbles can be seeded from a plasma following optical breakdown, by focusing an intense laser in water. The fast dynamics are associated with extreme states of gas and liquid, especially in the nascent state. This offers a unique setting to probe water and water vapor far-from equilibrium. However, current optical techniques cannot quantify these early states due to contrast and resolution limitations. X-ray holography with single X-ray free-electron laser pulses has now enabled a quasi-instantaneous high resolution structural probe with contrast proportional to the electron density of the object. In this work, we demonstrate cone-beam holographic flash imaging of laser-induced cavitation bubbles in water with nanofocused X-ray free-electron laser pulses. We quantify the spatial and temporal pressure distribution of the shockwave surrounding the expanding cavitation bubble at time delays shortly after seeding and compare the results to numerical simulations.

The cochlea of our auditory system is an intricate structure deeply embedded in the temporal bone. Compared with other sensory organs such as the eye, the cochlea has remained poorly accessible for investigation, for example, by imaging. This limitation also concerns the further development of technology for restoring hearing in the case of cochlear dysfunction, which requires quantitative information on spatial dimensions and the sensorineural status of the cochlea. Here, we employed X-ray phase-contrast tomography and light-sheet fluorescence microscopy and their combination for multiscale and multimodal imaging of cochlear morphology in species that serve as established animal models for auditory research. We provide a systematic reference for morphological parameters relevant for cochlear implant development for rodent and nonhuman primate models. We simulate the spread of light from the emitters of the optical implants within the reconstructed nonhuman primate cochlea, which indicates a spatially narrow optogenetic excitation of spiral ganglion neurons., Competing Interests: Competing interest statement: T.M. and D.K. are co-founders of OptoGenTech company.

Background: Enteric neuropathy is described in most patients with gastrointestinal dysmotility and may be found together with reduced intraepidermal nerve fiber density (IENFD). The aim of this pilot study was to assess whether three-dimensional (3d) imaging of skin biopsies could be used to examine various tissue components in patients with gastrointestinal dysmotility., Material and Methods: Four dysmotility patients of different etiology and two healthy volunteers were included. From each subject, two 3-mm punch skin biopsies were stained with antibodies against protein gene product 9.5 or evaluated as a whole with two X-ray phase-contrast computed tomography (CT) setups, a laboratory µCT setup and a dedicated synchrotron radiation nanoCT end-station., Results: Two patients had reduced IENFD, and two normal IENFD, compared with controls. µCT and X-ray phase-contrast holographic nanotomography scanned whole tissue specimens, with optional high-resolution scans revealing delicate structures, without differentiation of various fibers and cells. Irregular architecture of dermal fibers was observed in the patient with Ehlers-Danlos syndrome and the patient with idiopathic dysmotility showed an abundance of mesenchymal ground substance., Conclusions: 3d phase-contrast tomographic imaging may be useful to illustrate traits of connective tissue dysfunction in various organs and to demonstrate whether disorganized dermal fibers could explain organ dysfunction., (© 2020 The Authors. Skin Research and Technology published by John Wiley & Sons Ltd.)

Single-pulse holographic imaging at XFEL sources with 10 12 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., (open access.)

Small unilamellar vesicles (20-100 nm diameter) are model systems for strongly curved lipid membranes, in particular for cell organelles. Routinely, small-angle X-ray scattering (SAXS) is employed to study their size and electron-density profile (EDP). Current SAXS analysis of small unilamellar vesicles (SUVs) often employs a factorization into the structure factor (vesicle shape) and the form factor (lipid bilayer electron-density profile) and invokes additional idealizations: (i) an effective polydispersity distribution of vesicle radii, (ii) a spherical vesicle shape and (iii) an approximate account of membrane asymmetry, a feature particularly relevant for strongly curved membranes. These idealizations do not account for thermal shape fluctuations and also break down for strong salt- or protein-induced deformations, as well as vesicle adhesion and fusion, which complicate the analysis of the lipid bilayer structure. Presented here are simulations of SAXS curves of SUVs with experimentally relevant size, shape and EDPs of the curved bilayer, inferred from coarse-grained simulations and elasticity considerations, to quantify the effects of size polydispersity, thermal fluctuations of the SUV shape and membrane asymmetry. It is observed that the factorization approximation of the scattering intensity holds even for small vesicle radii (∼30 nm). However, the simulations show that, for very small vesicles, a curvature-induced asymmetry arises in the EDP, with sizeable effects on the SAXS curve. It is also demonstrated that thermal fluctuations in shape and the size polydispersity have distinguishable signatures in the SAXS intensity. Polydispersity gives rise to low- q features, whereas thermal fluctuations predominantly affect the scattering at larger q , related to membrane bending rigidity. Finally, it is shown that simulation of fluctuating vesicle ensembles can be used for analysis of experimental SAXS curves., (© Veronica Chappa et al. 2021.)