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Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently-labelled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods such as synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) or serial blockface scanning electron microscopy (SBF-SEM) might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging.Here we showcase the utility of a novel femtosecond laser device to prepare microscopic samples (1) of an optimised geometry for synchrotron X-ray microscopy as well as (2) for subsequent volume electron microscopy applications, embedded in a wider correlative multimodal imaging workflow (Fig. 1).

Like the vertebrate spinal cord, the insect ventral nerve cord (VNC) mediates limb sensation and motor control. Here, we applied automated tools for electron microscopy (EM) volume alignment, neuron reconstruction, and synapse prediction to create a draft connectome of theDrosophilaVNC. To interpret the VNC connectome, it is crucial to know its relationship with the rest of the body. We therefore mapped the muscle targets of leg and wing motor neurons in the connectome by comparing their morphology to genetic driver lines, dye fills, and x-ray holographic nano-tomography volumes of the fly leg and wing. Knowing the outputs of the connectome allowed us to identify neural circuits that coordinate the wings with the middle and front legs during escape takeoff. We provide the draft VNC connectome and motor neuron atlas, along with tools for programmatic and interactive access, as community resources.

Our ability to sense and move our bodies relies on proprioceptors, sensory neurons that detect mechanical forces within the body. Proprioceptors are diverse: different subtypes detect different features of joint kinematics, such as position, directional movement, and vibration. However, because they are located within complex and dynamic peripheral tissues, the underlying mechanisms of proprioceptor feature selectivity remain poorly understood. Here, we investigate molecular and biomechanical contributions to proprioceptor diversity in theDrosophilaleg. Using single-nucleus RNA sequencing, we found that different proprioceptor subtypes express similar complements of mechanosensory and other ion channels. However, anatomical reconstruction of the proprioceptive organ and connected tendons revealed major biomechanical differences between proprioceptor subtypes. We constructed a computational model of the proprioceptors and tendons, which identified a putative biomechanical mechanism for joint angle selectivity. The model also predicted the existence of a goniotopic map of joint angle among position-tuned proprioceptors, which we confirmed using calcium imaging. Our findings suggest that biomechanical specialization is a key determinant of proprioceptor feature selectivity inDrosophila. More broadly, our discovery of proprioceptive maps in the fly leg reveals common organizational principles between proprioception and other topographically organized sensory systems.

Living organisms form a large variety of hierarchically structured extracellular functional tissues. Remarkably, these materials exhibit regularity and structural coherence across multiple length scales, far beyond the size of a single cell. Here, synchrotron-based nanotomographic imaging in combination with machine-learning-based segmentation is used to reveal the structural synchronization process of nacre forming in the shell of the mollusc Unio pictorum. We show that the emergence of this highly regular layered structure is driven by a disorder-to-order transition achieved through the motion and interaction of screw-like structural dislocations with an opposite topological sign. Using an analogy to similar processes observed in liquid-crystalline systems, we demonstrate that these microstructural faults act as dissipative topological defects coupled by an elastic distortion field surrounding their cores. Their mutual annihilation results in structural synchronization that is simulated using the classical Kuramoto model. The developed experimental, theoretical and numerical framework provides a comprehensive physical view of the formation of biogenic materials. Molluscs assemble layers of material in the shells around them with a high level of control. Here the authors observe the structural evolution of layer formation and propose a mechanism reminiscent of topological defect dynamics in liquid crystals.

Significance Axons, the brain’s communication cables, have been described as cylinders since their discovery in 1860. Their structure is linked to how fast they conduct signals and is thus indicative of brain health and function. Here, we demonstrate an interplay between the micromorphology of axons and other extra-axonal structures, showing that axons are noncylindrical and exhibit environment-dependent diameter and trajectory variations. The nonspecificity in diameter, and thus conduction velocity, challenges the current knowledge of how axons communicate signals. Diffusion magnetic resonance imaging can be used to measure axon diameter in the living brain in order to explore the brain network and detect potential biomarkers of disease, but we show here that the observed complex morphologies of axons bias these measurements., Axonal conduction velocity, which ensures efficient function of the brain network, is related to axon diameter. Noninvasive, in vivo axon diameter estimates can be made with diffusion magnetic resonance imaging, but the technique requires three-dimensional (3D) validation. Here, high-resolution, 3D synchrotron X-ray nano-holotomography images of white matter samples from the corpus callosum of a monkey brain reveal that blood vessels, cells, and vacuoles affect axonal diameter and trajectory. Within single axons, we find that the variation in diameter and conduction velocity correlates with the mean diameter, contesting the value of precise diameter determination in larger axons. These complex 3D axon morphologies drive previously reported 2D trends in axon diameter and g-ratio. Furthermore, we find that these morphologies bias the estimates of axon diameter with diffusion magnetic resonance imaging and, ultimately, impact the investigation and formulation of the axon structure–function relationship.

There is a growing interest in developing 3D microscopy for the exploration of thick biological tissues. Recently, 3D X-ray nanocomputerised tomography has proven to be a suitable technique for imaging the bone lacunocanalicular network. This interconnected structure is hosting the osteocytes which play a major role in maintaining bone quality through remodelling processes. 3D images have the potential to reveal the architecture of cellular networks, but their quantitative analysis remains a challenge due to the density and complexity of nanometre sized structures and the need to handle and process large datasets, for example, 20483 voxels corresponding to 32 GB per individual image in our case. In this work, we propose an efficient image processing approach for the segmentation of the network and the extraction of characteristic parameters describing the 3D structure. These parameters include the density of lacunae, the porosity of lacunae and canaliculi, and morphological features of lacunae (volume, surface area, lengths, anisotropy etc.). We also introduce additional parameters describing the local environment of each lacuna and its canaliculi. The method is applied to analyse eight human femoral cortical bone samples imaged by magnified X-ray phase nanotomography with a voxel size of 120 nm, which was found to be a good compromise to resolve canaliculi while keeping a sufficiently large field of view of 246 μm in 3D. The analysis was performed on a total of 2077 lacunae showing an average length, width and depth of 17.1 μm × 9.2 μm × 4.4 μm, with an average number of 58.2 canaliculi per lacuna and a total lacuno-canalicular porosity of 1.12%. The reported descriptive parameters provide information on the 3D organisation of the lacuno-canalicular network in human bones.

Purpose Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. Methods We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer’s disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. Results In 3xTgAD mice, the observed hyperdensity was identified as amyloid-β and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. Conclusions This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer’s disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors.

To investigate the performance of three-dimensional (3D) nanostructures, it is vital to study their internal structure with a methodology that keeps the device fully functional and ready for further integration. To this aim, we introduce here traceless X-ray tomography (TXT) that combines synchrotron X-ray holographic tomography with high X-ray photon energies (17 keV) in order to study nanostructures "as is" on massive silicon substrates. The combined strengths of TXT are a large total sample size to field-of-view ratio and a large penetration depth. We study exemplary 3D photonic band gap crystals made by CMOS-compatible means and obtain real space 3D density distributions with 55 nm spatial resolution. TXT identifies why nanostructures that look similar in electron microscopy have vastly different nanophotonic functionality: one "good" crystal with a broad photonic gap reveals 3D periodicity as designed; a second "bad" structure without a gap reveals a buried void, and a third "ugly" one without gap is shallow due to fabrication errors. Thus, TXT serves to nondestructively differentiate between the possible reasons of not finding the designed and expected performance and is therefore a powerful tool to critically assess 3D functional nanostructures.

In an article examining the physiology of Early Jurassic mammaliaform stem-mammals, we used proxies for basal and maximum metabolic rate, providing evidence that two key fossil mammaliaforms had metabolic rates closer to modern reptiles than modern mammals1. Meiri and Levin2 questioned the use of our proxy for basal metabolic rate – terrestrial species maximum lifespan in the wild. Here, we explore the evidence behind these differences in viewpoint, and rebut specific points raised by these authors.

International audience; Benefiting from the recent advances of synchrotron X-ray nanoprobes, we demonstrate three-dimensional (3D) correlative nano-imaging on malaria-infected human red blood cells. By combining X-ray fluorescence to-mography and phase contrast nanotomography on the same cell with sub-100 nm pixel size, we establish a routine workflow from the data acquisition, data processing to tomographic reconstruction. We quantitatively compare the elemental volumes obtained with different reconstruction methods, the total variation minimization giving the most satisfactory results. We reveal elemental correlations in different cell compartments more reliably on reconstructions as opposed to 2D projections. Finally, we determine for the first time the 3D mass fraction maps of multiple elements at sub-cellular level. The estimated total number of Fe atoms and the total mass of the red blood cell show very good agreement with previously reported values.

Bone is an intriguingly complex material. It combines high strength, toughness and lightweight via an elaborate hierarchical structure. This structure results from a biologically driven self-assembly and self-organisation, and leads to different deformation mechanisms along the length scales. Characterising multiscale bone mechanics is fundamental to better understand these mechanisms including changes due to bone-related diseases. It also guides us in the design of new bio-inspired materials. A key-gap in understanding bone’s behaviour exists for its fundamental mechanical unit, the mineralised collagen fibre, a composite of organic collagen molecules and inorganic mineral nanocrystals. Here, we report an experimentally informed statistical elasto-plastic model to explain the fibre behaviour including the nanoscale interplay and load transfer with its main mechanical components. We utilise data from synchrotron nanoscale imaging, and combined micropillar compression and synchrotron X-ray scattering to develop the model. We see that a 10-15% micro- and nanomechanical heterogeneity in mechanical properties is essential to promote the ductile microscale behaviour preventing an abrupt overall failure even when individual fibrils have failed. We see that mineral particles take up 45% of strain compared to collagen molecules while interfibrillar shearing seems to enable the ductile post-yield behaviour. Our results suggest that a change in mineralisation and fibril-to-matrix interaction leads to different mechanical properties among mineralised tissues. Our model operates at crystalline-, molecular- and continuum-levels and sheds light on the micro- and nanoscale deformation of fibril-matrix reinforced composites.

Attributingin vivoneurophysiology to the brains’ ultrastructure requires a large field of view containing contextual anatomy. Electron microscopy (EM) is the gold standard technique to identify ultrastructure, yet acquiring volumes containing full mammalian neural circuits is challenging and time consuming using EM. Here, we show that synchrotron X-ray computed tomography (SXRT) provides rapid imaging of EM-prepared tissue volumes of several cubic millimetres. Resolution was sufficient for distinguishing cell bodies as well as for tracing apical dendrites in olfactory bulb and hippocampus, for up to 350 μm. Correlating EM with SXRT allowed us to associate dendritic spines on pyramidal cell apical dendrites in the stratum radiatum to their corresponding soma locations. Superficial pyramidal neurons had larger spine apparatus density compared to deeper ones, implying differential synaptic plasticity for superficial and deeper cells. Finally, we show that X-ray tomography and volume EM can be reliably correlated to priorin vivoimaging. Thus, combining functional measurements with multiscale X-ray microscopy and volume EM establishes a correlative workflow that enables functional and structural investigation of subcellular features in the context of cellular morphologies, tissues and ultimately whole organs.

X-ray Phase-contrast nano-tomography and X-ray fluorescence microscopy was performed on modern Pinaceae (pine) pollen at the ID16A nano-imaging beamline of the European Synchrotron (ESRF), in France.

Despite considerable advances in knowledge of the anatomy, ecology and evolution of early mammals, far less is known about their physiology. Evidence is contradictory concerning the timing and fossil groups in which mammalian endothermy arose. To determine the state of metabolic evolution in two of the earliest stem-mammals, the Early Jurassic Morganucodon and Kuehneotherium, we use separate proxies for basal and maximum metabolic rate. Here we report, using synchrotron X-ray tomographic imaging of incremental tooth cementum, that they had maximum lifespans considerably longer than comparably sized living mammals, but similar to those of reptiles, and so they likely had reptilian-level basal metabolic rates. Measurements of femoral nutrient foramina show Morganucodon had blood flow rates intermediate between living mammals and reptiles, suggesting maximum metabolic rates increased evolutionarily before basal metabolic rates. Stem mammals lacked the elevated endothermic metabolism of living mammals, highlighting the mosaic nature of mammalian physiological evolution., Modern mammals are endothermic, but it has not been clear when this type of metabolism evolved. Here, Newham et al. analyse tooth and bone structure in Early Jurassic stem-mammal fossils to estimate lifespan and blood flow rates, which inform about basal and maximum metabolic rates, respectively, and show these stem-mammals had metabolic rates closer to modern ectothermic reptiles than to endothermic mammals.

X-ray microscopy is increasingly used in biology, but in most cases only in a qualitative way. We present here a 3D correlative cryo X-ray microscopy approach suited for the quantification of molar concentrations and structure in native samples at nanometer scale. The multimodal approach combines X-ray fluorescence and X-ray holographic nanotomography on "thick" frozen-hydrated cells. The quantitativeness of the X-ray fluorescence reconstruction is improved by estimating the self-attenuation from the 3D holography reconstruction. Applied to complex macrophage cells, we extract the quantification of major and minor elements heavier than phosphorus, as well as the density, in the different organelles. The intracellular landscape shows remarkable elemental differences. This novel analytical microscopy approach will be of particular interest to investigate complex biological and chemical systems in their native environment.

Recently, increasing attention has been given to the study of osteocytes, the cells that are thought to play an important role in bone remodeling and in the mechanisms of bone fragility. The interconnected osteocyte system is deeply embedded inside the mineralized bone matrix and lies within a closely fitted porosity known as the lacuno-canalicular network. However, quantitative data on human samples remain scarce, mostly measured in 2D, and there are gaps to be filled in terms of spatial resolution. In this work, we present data on femoral samples from female donors imaged with isotropic 3D spatial resolution by magnified X-ray phase nano computerized-tomography. We report quantitative results on the 3D structure of canaliculi in human femoral bone imaged with a voxel size of 30 nm. We found that the lacuno-canalicular porosity occupies on average 1.45% of the total tissue volume, the ratio of the canalicular versus lacunar porosity is about 37.7%, and the primary number of canaliculi stemming from each lacuna is 79 on average. The examination of this number at different distances from the surface of the lacunae demonstrates branching in the canaliculi network. We analyzed the impact of spatial resolution on quantification by comparing parameters extracted from the same samples imaged with 120 nm and 30 nm voxel sizes. To avoid any bias related to the analysis region, the volumes at 120 nm and 30 nm were registered and cropped to the same field of view. Our results show that the measurements at 120 and 30 nm are strongly correlated in our data set but that the highest spatial resolution provides more accurate information on the canaliculi network and its branching properties.

A deeper knowledge of the architecture of the peripheral nerve with three-dimensional (3D) imaging of the nerve tissue at the sub-cellular scale may contribute to unravel the pathophysiology of neuropathy. Here we demonstrate the feasibility of X-ray phase contrast holographic nanotomography to enable 3D imaging of nerves at high resolution, while covering a relatively large tissue volume. We show various subcomponents of human peripheral nerves in biopsies from patients with type 1 and 2 diabetes and in a healthy subject. Together with well-organized, parallel myelinated nerve fibres we show regenerative clusters with twisted nerve fibres, a sprouted axon from a node of Ranvier and other specific details. A novel 3D construction (with movie created) of a node of Ranvier with end segment of a degenerated axon and sprout of a regenerated one is captured. Many of these architectural elements are not described in the literature. Thus, X-ray phase contrast holographic nanotomography enables identifying specific morphological structures in 3D in peripheral nerve biopsies from a healthy subject and from patients with type 1 and 2 diabetes. Funding Agencies|EU InterReg project MAX4ESSFUN [KU-008]; Swedish Research CouncilSwedish Research Council [E0605401, E0605402]; Lund University

New scientific frontiers in biomedicine, materials science, and nanotechnology make increasing use of characterization methods at the mesoscopic and nanometer scale. These studies of heterogeneous ...

X-ray phase tomography aims at reconstructing the 3D electron density distribution of an object. It offers enhanced sensitivity compared to attenuation-based X-ray absorption tomography. In propagation-based methods, phase contrast is achieved by letting the beam propagate after interaction with the object. The phase shift is then retrieved at each projection angle, and subsequently used in tomographic reconstruction to obtain the refractive index decrement distribution, which is proportional to the electron density. Accurate phase retrieval is achieved by combining images at different propagation distances. For reconstructions of good quality, the phase-contrast images recorded at different distances need to be accurately aligned. In this work, we characterise the artefacts related to misalignment of the phase-contrast images, and investigate the use of different registration algorithms for aligning in-line phase-contrast images. The characterisation of artefacts is done by a simulation study and comparison with experimental data. Loss in resolution due to vibrations is found to be comparable to attenuation-based computed tomography. Further, it is shown that registration of phase-contrast images is nontrivial due to the difference in contrast between the different images, and the often periodical artefacts present in the phase-contrast images if multilayer X-ray optics are used. To address this, we compared two registration algorithms for aligning phase-contrast images acquired by magnified X-ray nanotomography: one based on cross-correlation and one based on mutual information. We found that the mutual information-based registration algorithm was more robust than a correlation-based method.

Significance Silver nanowires are a type of novel fiber that are likely to have wide application in consumer electronics but that can also carry risk for cell internalization and toxicity. Here, we show that silver nanowire toxicity can be greatly diminished by reducing nanowire diameter without affecting device performance. X-ray microscopy and supporting studies revealed that endolysosome membranes can mechanically crumple thin silver nanowires, the likely mechanism for diminishing toxicity., Fibrous particles interact with cells and organisms in complex ways that can lead to cellular dysfunction, cell death, inflammation, and disease. The development of conductive transparent networks (CTNs) composed of metallic silver nanowires (AgNWs) for flexible touchscreen displays raises new possibilities for the intimate contact between novel fibers and human skin. Here, we report that a material property, nanowire-bending stiffness that is a function of diameter, controls the cytotoxicity of AgNWs to nonimmune cells from humans, mice, and fish without deterioration of critical CTN performance parameters: electrical conductivity and optical transparency. Both 30- and 90-nm-diameter AgNWs are readily internalized by cells, but thinner NWs are mechanically crumpled by the forces imposed during or after endocytosis, while thicker nanowires puncture the enclosing membrane and release silver ions and lysosomal contents to the cytoplasm, thereby initiating oxidative stress. This finding extends the fiber pathology paradigm and will enable the manufacture of safer products incorporating AgNWs.