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Abstract The performance of machine learning algorithms, when used for segmenting 3D biomedical images, does not reach the level expected based on results achieved with 2D photos. This may be explained by the comparative lack of high-volume, high-quality training datasets, which require state-of-the-art imaging facilities, domain experts for annotation and large computational and personal resources. The HR-Kidney dataset presented in this work bridges this gap by providing 1.7 TB of artefact-corrected synchrotron radiation-based X-ray phase-contrast microtomography images of whole mouse kidneys and validated segmentations of 33 729 glomeruli, which corresponds to a one to two orders of magnitude increase over currently available biomedical datasets. The image sets also contain the underlying raw data, threshold- and morphology-based semi-automatic segmentations of renal vasculature and uriniferous tubules, as well as true 3D manual annotations. We therewith provide a broad basis for the scientific community to build upon and expand in the fields of image processing, data augmentation and machine learning, in particular unsupervised and semi-supervised learning investigations, as well as transfer learning and generative adversarial networks.

Abstract Achromatic doublets are combinations of two individual lenses designed to focus different wavelengths of light in the same position. Apochromatic optics are improved versions of the achromatic schemes which extend the wavelength range significantly. Both achromatic and apochromatic optics are well-established for visible light. However, X-ray achromatic lenses did not exist until very recently, and X-ray apochromatic lenses have never been experimentally demonstrated. Here, we create an X-ray apochromatic lens system using an appropriate combination of a Fresnel zone plate and a diverging compound refractive lens with a tuned separation distance. The energy-dependent performance of this apochromat was characterized at photon energies between 6.5 and 13.0 keV by ptychographic reconstruction of the focal spot and scanning transmission X-ray microscopy of a resolution test sample. The apochromat delivered a reconstructed focal spot size of 940 × 740 nm2. The apochromatic combination shows a four-fold improvement in the chromatic aberration correction range compared to an achromatic doublet configuration. Thus, apochromatic X-ray optics have the potential to increase the focal spot intensity for a wide variety of X-ray applications.

Macular edema (ME) remains a primary cause of visual deterioration in uveitis. Visual acuity (VA) can often be maintained using corticosteroid depot systems. This study evaluated the efficacy of a fluocinolone acetonide (FAc) intravitreal implant (ILUVIEN®) in treating non-infectious uveitis using real-world data. This retrospective analysis included 135 eyes subdivided into responders and non-responders. Central retinal thickness (CRT), VA, and intraocular pressure (IOP) were followed over time. A significant decrease in CRT and an increase in VA were observed in all eyes throughout the follow-up period (p < 0.01). An IOP increase (p = 0.028) necessitated treatment in 43% of eyes by Month 6. Non-responders were older (p = 0.004) and had been treated with more dexamethasone (DEX) implants (p = 0.04); 89.3% had a defect in the external limiting membrane (ELM) and inner/outer segment (IS/OS) zone (p < 0.001). Immunomodulatory therapy had no impact on treatment response. Pars plana vitrectomy (PPV) patients had a mean CRT reduction of 47.55 µm and a reduced effect by Month 24 (p = 0.046) versus non-PPV patients. We conclude that the FAc implant achieves long-term control of CRT and improves VA. Increases in IOP were manageable. Eyes with a previous PPV showed milder results. Data showed a correlation between older age, a damaged ELM and IS/OS zone, frequent DEX inserts, and poorer outcome measures.

External ecological interactions and intrinsic biological parameters affect evolutionary pathways and animal diversity. Here, the authors use ruminant inner ear morphology to investigate patterns of diversity through 33 million years, finding clade-dependent climate and paleogeographic trends.

Introduction: Advanced localized retinoblastoma can be cured by enucleation, but extraocular spread of retinoblastoma cells is associated with a high mortality. Risk-stratified adjuvant treatment with chemotherapy and radiotherapy has been shown to reduce the risk for extraocular relapse in children with histopathological risk factors. Methods: Data of 184 patients with retinoblastoma and primary enucleation were collected in a prospective, multicenter, observational study between 2013 and 2020. The clinical characteristics were evaluated as risk factors and progression-free and overall survival rates were compared. Results: Seventy-one percent of 184 children with retinoblastoma treated with primary enucleation were diagnosed with low risk histopathological factors (pT1/pT2a) and received no adjuvant therapy. Children with intermediate risk (pT2b,pT3; 48 children, 26.0%) and high risk for metastasis (pT4; 5 children, 2.7%) received risk-stratified adjuvant treatment. None of the children with low risk or intermediate risk (pT1-pT3) relapsed, but two of five children with high-risk retinoblastoma (pT4) developed extraocular relapses and one deceased. The 2-year progression-free survival rate and 2-year overall survival rate was 100% for children with pT1-3 retinoblastoma. However, the 2-year progression-free survival rate and 2-year overall survival rate for children with pT4 was statistically notably reduced with 2 of 5 children developing progression and 1 death among the 5 children within 2 years after diagnosis. Conclusion: Primary enucleation alone and with additional risk-stratified adjuvant chemotherapy treatment provides high cure rates in patients with pT1-3 retinoblastoma, but children with pT4 retinoblastoma remain at high risk to develop extraocular retinoblastoma. International prospective clinical trials are required to evaluate reduction of intensity of adjuvant chemotherapy in some risk groups (pT2, pT3) and intensification for pT4 retinoblastoma.

Nephrotoxicity is an important drug safety aspect to be assessed during drug discovery and development. To study renal toxicity, in vitro cell-based assays are often used. Unfortunately, translating the results of such cell assays to vertebrates including human remains challenging. Therefore, we aim to evaluate whether zebrafish larvae (ZFL) could serve as a vertebrate screening model to detect gentamicin-induced changes of kidney glomeruli and proximal tubules. To validate the model, we compared the results of ZFL with those obtained from kidney biopsies of gentamicin-treated mice. We used transgenic zebrafish lines expressing enhanced green fluorescent proteins in the glomerulus to visualize glomerular damage. Synchrotron radiation-based computed tomography (SRμCT) is a label-free approach providing three-dimensional representations of renal structures with micrometre resolution. Clinically used gentamicin concentrations induce nephrotoxicity and affect glomerular and proximal tubular morphology. Findings were confirmed in mice and ZFL. There was a strong correlation between fluorescent signals in ZFL, SRμCT- derived descriptors of glomerular and proximal tubular morphology and the histological analysis of mouse kidney biopsies. A combination of SRμCT and confocal microscopy provides unprecedented insights into anatomical structures of the zebrafish kidney. Based on our findings, we suggest to use ZFL as a predictive vertebrate screening model to study drug-induced nephrotoxicity and to bridge the gap between cell culture-based test systems and experiments in mammals.

Neuromodulation treatments are based on the functional interface between the brain and man-made electrodes. Chronic inflammation around the implant, however, occurs as a result of mechanical mismatching between soft brain tissue and the stiff metallic electrode. After water uptake, cellulose films should prevent encapsulation of the electrode. In this work, we reinforced cellulose films with silk fibre networks, improving tear strength by a factor of four to seven and maintaining flexibility, characterised by an elastic modulus between 100 and 200 MPa. Whereas sputtered gold on flat films exhibited restricted adhesion, micro-patterning guaranteed reasonable adhesion of nanometre-thin gold to cellulose substrates. Micro-patterned cellulose films coated in 80 nm-thin gold retained conductivity for strains as large as 30%, while sheet resistance increased by a factor of about 30. Fabrication of the biocompatible electrodes is efficient and compatible with large-scale production.

Distraction osteogenesis is a clinically established technique for lengthening, molding and shaping bone by new bone formation. The experimental evaluation of this expensive and time-consuming treatment is of high impact for better understanding of tissue engineering but mainly relies on a limited number of histological slices. These tissue slices contain two-dimensional information comprising only about one percent of the volume of interest. In order to analyze the soft and hard tissues of the entire jaw of a single rat in a multimodal assessment, we combined micro computed tomography (µCT) with histology. The µCT data acquired before and after decalcification were registered to determine the impact of decalcification on local tissue shrinkage. Identification of the location of the H&E-stained specimen within the synchrotron radiation-based µCT data collected after decalcification was achieved via non-rigid slice-to-volume registration. The resulting bi- and tri-variate histograms were divided into clusters related to anatomical features from bone and soft tissues, which allowed for a comparison of the approaches and resulted in the hypothesis that the combination of laboratory-based µCT before decalcification, synchrotron radiation-based µCT after decalcification and histology with hematoxylin-and-eosin staining could be used to discriminate between different types of collagen, key components of new bone formation.

Purpose. Diabetic macular edema (DME) is the most common cause of blindness in the working-age population. Spectral-domain optical coherence tomography (SD-OCT) allows detection and monitoring of the edema and a detailed analysis of the retinal structure. Hyperreflective foci (HF) are small, circumscribed lesions on OCT, and their origin is yet to be determined. Our study was aimed to shed light on HF pathophysiology, by analyzing their number and location in DME patients at baseline and after therapy. Methods. A prospective, observational study on 59 eyes of 51 DME patients who were treated with antivascular endothelial growth factor (VEGF) therapy (VEGF group, n=40 eyes) or dexamethasone implant (DEX group, n=19). HF and hard exudates (HE) were discriminated by their appearance on fundus photographs and their size on OCT. Quantity and location of HF and HE were analyzed at baseline and after therapy. Results. DME decreased in 75% of patients in the VEGF (455.5 μm vs. 380.8 μm, p=0.02) and in 95% of patients in the DEX group (471.6 μm vs. 381.9 μm, p=0.007). The number of foci decreased in 62.5% of patients after anti-VEGF (130.6 vs. 111.1, p=0.07) and in 68% of patients after dexamethasone injection ((123.4 vs. 94.9, p=0.02) 5.1). A subgroup of 15% of eyes, all treated with anti-VEGF, showed accumulation of larger HF in outer retinal layers to visible HE during DME resolution, whereas smaller HF, found in all retinal layers, remained unchanged. There was a trend towards a dynamic shift of the foci from inner to outer retinal layers. Conclusion. The dynamic rearrangement of the small HF and their slightly greater reduction after anti-inflammatory therapy suggest inflammatory cells as their origin, whereas larger HF in the outer retinal layers correspond to microexudates. Furthermore, we found a more favourable outcome in patients with HF after treatment with dexamethasone implants compared to anti-VEGF agents.

The human brain is one of the most fascinating and important structures in nature. So far, his-tology has been the gold standard for imaging anatomical features on the sub-cellular level. Us-ing standard optical microscopy spatial resolution is restricted to a fraction of a micron. Recip-rocal-space techniques, including small-angle X-ray scattering (SAXS), with an inverse relation-ship between a nanostructure's size and scattering angle, have been used to study animal and human tissues. The myelin sheaths responsible for insulating axons, for example, exhibit a pe-riodicity between 15 and 18 nm. To localize their abundance and orientation, we have performed SAXS measurements with a micrometer-sized beam along a slice of the human brain and related them to the histology of myelin-stained tissue. The experimental results elucidate that, compared to histology, micrometer resolved SAXS provides information about the orientation of myelin, and consequently, axons, in addition to myelin abundance. The acquired data show color-coded orientation maps of the nanostructures, thereby providing a detailed insight into a relevant part of the human brain's nano-anatomy.

Long-term interfacing with neural tissue is key for the diagnosis and therapy of neurological disorder. Coatings with dedicated micro- and nanostructures have been proposed, such as gold nanowires, platinum nanostructured by electrochemical roughening, columnar and porous titanium nitride, carbon nanotubes, and conductive polymers. The performance of these coatings, however, is limited because of the mechanical mismatch between implant and neural tissue. Herein, we show that micro- and nanostructured, soft and conductive elastomer films can be obtained by depositing gold on nanometer-thin thiol-functionalized polydimethylsiloxane (PDMS) films. Additionally, microstructured polyether ether ketone (PEEK) films enable directional ordering in topology. The formation of soft and conductive PDMS films with oriented wrinkles on the macroscopic scale was controlled by the ratio between the metal/elastomter thicknesses and the depth of thermally imprinted trenches. Four-point probe measurements revealed that the electrical conductivity is one order of magnitude higher than that of recently presented hydrogel formulations. Nano-indentations proved that the submicrometer-thin conductive elastomer exhibit an average elastic modulus well below 10 MPa. This material system can be made tens of micrometers thin, and, therefore, has the potential to address several challenges of current implantable neural interfaces for the central nervous system, e.g. fabrication of softer and more flexible micrometer-thin spinal cord arrays.

The stapes is the smallest bone of the mammalian skeleton. Being the innermost middle ear ossicle, it is in contact with the inner ear and is directly responsible for sound transmission into it. Today, Ruminantia are one of the most diversified groups of large mammals with more than 200 species. However, their stapes has been very little studied. Here we investigate the shape of 66 stapes from 44 species of extant and extinct Ruminantia, including intra-individual and intra-specific observations, based on 3D tomographic data. Shape differences and similarities are quantitatively discussed thanks to 3D geometric morphometrics. The overall size of the stapes scales with a negative allometry in comparison to body mass. Moreover, the overall shape of the stapes informs about phylogeny. A trend is observed from a concave posterior crus with an enlarged stapes capitulum in Antilocapridae to a relatively straight posterior crus with a little reduced stapes capitulum in Cervidae, Bovidae being intermediate. In addition, the stapes of Antilocapridae is relatively trapezoid in lateral view; that of Cervidae is more triangular in lateral view; and that of Bovidae is relatively rectangular in lateral view. The stapedial footplate shape also gives phylogenetic information. The Tragulidae stapedial footplate is antero-posteriorly asymmetrical. The stapedial footplate is ovoid in stem Pecora. It is asymmetrical in Bovidae, while it is more symmetrical in Cervidae. This is in agreement with previous studies on the ruminant bony labyrinth showing that the oval window, the counterpart of the stapedial footplate on the inner ear, bears a strong phylogenetic signal already distinguishable in early Miocene ruminants.

One major limitation for the vascularization of bone substitutes used for filling is the presence of mineral blocks. The newly-formed blood vessels are stopped or have to circumvent the mineral blocks, resulting in inefficient delivery of oxygen and nutrients to the implant. This leads to necrosis within the implant and to poor engraftment of the bone substitute. The aim of the present study is to provide a bone substitute currently used in the clinic with suitably guided vascularization properties. This therapeutic hybrid bone filling, containing a mineral and a polymeric component, is fortified with pro-angiogenic smart nano-therapeutics that allow the release of angiogenic molecules. Our data showed that the improved vasculature within the implant promoted new bone formation and that the newly-formed bone swapped the mineral blocks of the bone substitutes much more efficiently than in non-functionalized bone substitutes. Therefore, we demonstrated that our therapeutic bone substitute is an advanced therapeutical medicinal product, with great potential to recuperate and guide vascularization that is stopped by mineral blocks, and can improve the regeneration of critical-sized bone defects. We have also elucidated the mechanism to understand how the newly-formed vessels can no longer encounter mineral blocks and pursue their course of vasculature, giving our advanced therapeutical bone filling great potential to be used in many applications, by combining filling and nano-regenerative medicine that currently fall short because of problems related to the lack of oxygen and nutrients.

Atherosclerosis gives rise to blood vessel occlusion associated with blood flow alteration and substantial increase of average wall shear stress. This modification was proved acting as a purely physical trigger for targeted vasodilator release from a particular type of liposomes composed of 1,3-diaminophospholipids (Pad-PC-Pad). The flow-induced structural changes of these faceted liposomes, however, are completely unknown. Therefore, spatially resolved small-angle X-ray scattering was combined with microfluidics to uniquely study the purely physical mechanisms, which give rise to the highly efficient drug release from mechanoresponsive liposomes of nanometer size. The microfluidic device, designed to mimic a stenotic blood vessel, consisted of a 1-mm-wide channel with a constriction, 125 ​μm in diameter. Here, the changes of the average bilayer thickness and the mean size of the mechanoresponsive liposomes have been locally detected under flow conditions. Overall shape and bilayer thickness do change already near the constriction inlet, but the alteration is dominant near the outlet. At a flow rate of 0.2 ​μL/s, the liposome's bilayer thickness increased by 30% compared to the situation well before the constriction and under static condition. The detected bilayer thickness increase of the faceted liposomes is in line with the mechanically induced loss of interdigitation between the phospholipid amide chains. These results imply that rather the gradient force than the wall shear stress provokes structural changes of Pad-PC-Pad liposomes and the related drug release at stenoses. The approach, i.e. the combination of microfluidics and spatially resolved small-angle X-ray scattering, paves the way to design highly efficient and specific systems for the targeted drug delivery at constrictions with predefined morphology.

Abstract There have been great efforts on the nanoscale 3D probing of brain tissues to image subcellular morphologies. However, limitations in terms of tissue coverage, anisotropic resolution, stain dependence, and complex sample preparation all hinder achieving a better understanding of the human brain functioning in the subcellular context. Herein, X‐ray nanoholotomography is introduced as an emerging synchrotron radiation‐based technology for large‐scale, label‐free, direct imaging with isotropic voxel sizes down to 25 nm, exhibiting a spatial resolution down to 88 nm. The procedure is nondestructive as it does not require physical slicing. Hence, it allows subsequent imaging by complementary techniques, including histology. The feasibility of this 3D imaging approach is demonstrated on human cerebellum and neocortex specimens derived from paraffin‐embedded tissue blocks. The obtained results are compared to hematoxylin and eosin stained histological sections and showcase the ability for rapid hierarchical neuroimaging and automatic rebuilding of the neuronal architecture at the level of a single cell nucleolus. The findings indicate that nanoholotomography can complement microscopy not only by large isotropic volumetric data but also by morphological details on the sub‐100 nm level, addressing many of the present challenges in brain tissue characterization and probably becoming an important tool in nanoanatomy.

Liposomal drug delivery systems can protect pharmaceutical substances and control their release. Systemic administration of liposomes, however, often activate the innate immune system, resulting in hypersensitivity reactions. These pseudo-allergic reactions can be interpreted as activating the complement system. Complement activation destroys and eliminates foreign substances, either directly through opsonization and the formation of the membrane attack complex (MAC), or by activating leukocytes and initiating inflammatory responses via mediators, such as cytokines. In this study, we investigated the in vitro immune toxicity of the recently synthesized Rad-PC-Rad liposomes, analyzing the liposome-induced complement activation. In five human sera, Rad-PC-Rad liposomes did not induce activation, but in one serum high sensitivity via alternative pathway was detected. Such a behavior in adverse phenomena is characteristic for patient-to-patient variation and, thus, the number of donors should be in the order of hundreds rather than tens, hence the present study based on six donors is preliminary. In order to further prove the suitability of mechano-responsive Rad-PC-Rad liposomes for clinical trials, the production of pro-inflammatory cytokines was examined by human white blood cells. The concentrations of the pro-inflammatory cytokines, IL-6, IL-12p70, TNF- α, and IL-1 β, induced by Rad-PC-Rad liposomal formulations, incubated with whole blood samples, were smaller or comparable to PBS (negative control). Because of this favorable in vitro hemocompatibility, in vivo investigations using these mechano-responsive liposomes should be designed.

Visualizing the internal architecture of large soft tissue specimens within the laboratory environment in a label-free manner is challenging, as the conventional absorption-contrast tomography yields a poor contrast. In this communication, we present the integration of an X-ray double-grating interferometer (XDGI) into an advanced, commercially available micro computed tomography system nanotom® m with a transmission X-ray source and a micrometer-sized focal spot. The performance of the interferometer is demonstrated by comparing the registered three-dimensional images of a human knee joint sample in phase- and conventional absorption-contrast modes. XDGI provides enough contrast (1.094 ± 0.152) to identify the cartilage layer, which is not recognized in the conventional mode (0.287 ± 0.003). Consequently, the two modes are complementary, as the present XDGI set-up only reaches a spatial resolution of (73 ± 6) μm, whereas the true micrometer resolution in the absorption-contrast mode has been proven. By providing complimentary information, XDGI is especially a supportive quantitative method for imaging soft tissues and visualizing weak X-ray absorbing species in the direct neighborhood of stronger absorbing components at the microscopic level.

Micro computed tomography has been combined with dedicated data analysis for the in vitro quantification of sub-surface enamel lesion mineralization. Two artificial white spot lesions, generated on a human molar crown in vitro, were examined. One lesion was treated with a self-assembling peptide intended to trigger nucleation of hydroxyapatite crystals. We non-destructively determined the local X-ray attenuation within the specimens before and after treatment. The three-dimensional data was rigidly registered. Three interpolation methods, i.e., nearest neighbor, tri-linear, and tri-cubic interpolation were evaluated. The mineralization of the affected regions was quantified via joint histogram analysis, i.e., a voxel-by-voxel comparison of the tomography data before and after mineralization. After ten days incubation, the mean mineralization coefficient reached 35.5% for the peptide-treated specimen compared to 11.5% for the control. This pilot study does not give any evidence for the efficacy of peptide treatment nor allows estimating the necessary number of specimens to achieve significance, but shows a sound methodological approach on the basis of the joint histogram analysis.

The tailoring of molecular weight distribution and the functional group density of vinyl-terminated polydimethylsiloxane (PDMS) by molecular beam deposition is demonstrated herein. Thermally evaporated PDMS and its residue are characterized using gel permeation chromatography and nuclear magnetic resonance. Thermal fragmentation of vinyl groups occurs for evaporation temperatures above 487 K (214 °C). At a background pressure of 10−6 mbar, the maximum molecular weight distribution is adjusted from (700 ± 100) g/mol to (6100 ± 100) g/mol with a polydispersity index of 1.06 ± 0.02. The content of vinyl-termination per repeating unit of PDMS is tailored from (2.8 ± 0.2)% to (5.6 ± 0.1)%. Molecular weights of vinyl-terminated PDMS evaporated at temperatures above 388 K (115 °C) correspond to those attributed to trimethyl-terminated PDMS. Side groups of linear PDMS dominate intermolecular interactions and vapor pressure.

Radiography has long been the standard approach to characterize carious lesions. Spatially resolved X-ray diffraction, specifically small-angle X-ray scattering (SAXS), has recently been applied to caries research. The aims of this combined SAXS and micro computed tomography (µCT) study were to locally characterize and compare the micro- and nanostructures of one natural carious lesion and of one artificially induced enamel lesion; and demonstrate the feasibility of an automated approach to combined SAXS and µCT data in segmenting affected and unaffected enamel. Enamel, demineralized by natural or artificial caries, exhibits a significantly reduced X-ray attenuation compared to sound enamel and gives rise to a drastically increased small-angle scattering signal associated with the presence of nanometer-size pores. In addition, X-ray scattering allows the assessment of the overall orientation and the degree of anisotropy of the nanostructures present. Subsequent to the characterization with µCT, specimens were analyzed using synchrotron radiation-based SAXS in transmission raster mode. The bivariate histogram plot of the projected data combined the local scattering signal intensity with the related X-ray attenuation from µCT measurements. These histograms permitted the segmentation of anatomical features, including the lesions, with micrometer precision. The natural and artificial lesions showed comparable features, but they also exhibited size and shape differences. The clear identification of the affected regions and the characterization of their nanostructure allow the artificially induced lesions to be verified against selected natural carious lesions, offering the potential to optimize artificial demineralization protocols. Analysis of joint SAXS and µCT histograms objectively segmented sound and affected enamel.

While calcium phosphate–based ceramics are currently the most widely used materials in bone repair, they generally lack tensile strength for initial load bearing. Bulk titanium is the gold standard of metallic implant materials, but does not match the mechanical properties of the surrounding bone, potentially leading to problems of fixation and bone resorption. As an alternative, nickel–titanium alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone better than any other metallic material. With the ultimate goal of fabricating porous implants for spinal, orthopedic and dental applications, nickel–titanium substrates were fabricated by means of selective laser melting. The response of human mesenchymal stromal cells to the nickel–titanium substrates was compared to mesenchymal stromal cells cultured on clinically used titanium. Selective laser melted titanium as well as surface-treated nickel–titanium and titanium served as controls. Mesenchymal stromal cells had similar proliferation rates when cultured on selective laser melted nickel–titanium, clinically used titanium, or controls. Osteogenic differentiation was similar for mesenchymal stromal cells cultured on the selected materials, as indicated by similar gene expression levels of bone sialoprotein and osteocalcin. Mesenchymal stromal cells seeded and cultured on porous three-dimensional selective laser melted nickel–titanium scaffolds homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold’s pore volume with extracellular matrix. The combination of bone-related mechanical properties of selective laser melted nickel–titanium with its cytocompatibility and support of osteogenic differentiation of mesenchymal stromal cells highlights its potential as a superior bone substitute as compared to clinically used titanium.