Multiscale photonic imaging of the native and implanted cochlea.

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.

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

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

Three-dimensional virtual histology of human cerebellum by X-ray phase-contrast tomography.

To quantitatively evaluate brain tissue and its corresponding function, knowledge of the 3D cellular distribution is essential. The gold standard to obtain this information is histology, a destructive and labor-intensive technique where the specimen is sliced and examined under a light microscope, providing 3D information at nonisotropic resolution. To overcome the limitations of conventional histology, we use phase-contrast X-ray tomography with optimized optics, reconstruction, and image analysis, both at a dedicated synchrotron radiation endstation, which we have equipped with X-ray waveguide optics for coherence and wavefront filtering, and at a compact laboratory source. As a proof-of-concept demonstration we probe the 3D cytoarchitecture in millimeter-sized punches of unstained human cerebellum embedded in paraffin and show that isotropic subcellular resolution can be reached at both setups throughout the specimen. To enable a quantitative analysis of the reconstructed data, we demonstrate automatic cell segmentation and localization of over 1 million neurons within the cerebellar cortex. This allows for the analysis of the spatial organization and correlation of cells in all dimensions by borrowing concepts from condensed-matter physics, indicating a strong short-range order and local clustering of the cells in the granular layer. By quantification of 3D neuronal "packing," we can hence shed light on how the human cerebellum accommodates 80% of the total neurons in the brain in only 10% of its volume. In addition, we show that the distribution of neighboring neurons in the granular layer is anisotropic with respect to the Purkinje cell dendrites.

Correlative x-ray phase-contrast tomography and histology of human brain tissue affected by Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder which is characterized by increasing dementia. It is accompanied by the development of extracellular ß-amyloid plaques and neurofibrillary tangles in the gray matter of the brain. Histology is the gold standard for the visualization of this pathology, but also has intrinsic shortcomings. Fully three-dimensional analysis and quantitative metrics of alterations in the tissue structure require a complementary approach. In this work we use x-ray phase-contrast tomography to obtain three-dimensional reconstructions of human hippocampal tissue affected by AD. Due to intrinsic electron density differences, tissue components and structures such as the granule cells of the dentate gyrus, blood vessels, or mineralized plaques can be identified and segmented in large volumes. Based on correlative histology, protein (tau, ß-amyloid) and elemental content (iron, calcium) can be attributed to certain morphological features occurring in the entire volume. In the vicinity of senile plaques, an accumulation of microglia in combination with a loss of neuronal cells can be observed.

A phase-retrieval toolbox for X-ray holography and tomography.

Propagation-based phase-contrast X-ray imaging is by now a well established imaging technique, which - as a full-field technique - is particularly useful for tomography applications. Since it can be implemented with synchrotron radiation and at laboratory micro-focus sources, it covers a wide range of applications. A limiting factor in its development has been the phase-retrieval step, which was often performed using methods with a limited regime of applicability, typically based on linearization. In this work, a much larger set of algorithms, which covers a wide range of cases (experimental parameters, objects and constraints), is compiled into a single toolbox - the HoloTomoToolbox - which is made publicly available. Importantly, the unified structure of the implemented phase-retrieval functions facilitates their use and performance test on different experimental data.

Axonal Ensheathment in the Nervous System of Lamprey: Implications for the Evolution of Myelinating Glia.

In the nervous system, myelination of axons enables rapid impulse conduction and is a specialized function of glial cells. Myelinating glia are the last cell type to emerge in the evolution of vertebrate nervous systems, presumably in ancient jawed vertebrates (gnathostomata) because jawless vertebrates (agnathans) lack myelin. We have hypothesized that, in these unmyelinated species, evolutionary progenitors of myelinating cells must have existed that should still be present in contemporary agnathan species. Here, we used advanced electron microscopic techniques to reveal axon-glia interactions in the sea lamprey Petromyzon marinus By quantitative assessment of the spinal cord and the peripheral lateral line nerve, we observed a marked maturation-dependent growth of axonal calibers. In peripheral nerves, all axons are ensheathed by glial cells either in bundles or, when larger than the threshold caliber of 3 µm, individually. The ensheathing glia are covered by a basal lamina and express SoxE-transcription factors, features of mammalian Remak-type Schwann cells. In larval lamprey, the ensheathment of peripheral axons leaves gaps that are closed in adults. CNS axons are also covered to a considerable extent by glial processes, which contain a high density of intermediate filaments, glycogen particles, large lipid droplets, and desmosomes, similar to mammalian astrocytes. Indeed, by in situ hybridization, these glial cells express the astrocyte marker Aldh1l1 Specimens were of unknown sex. Our observations imply that radial sorting, ensheathment, and presumably also metabolic support of axons are ancient functions of glial cells that predate the evolutionary emergence of myelin in jawed vertebrates.SIGNIFICANCE STATEMENT We used current electron microscopy techniques to examine axon-glia units in a nonmyelinated vertebrate species, the sea lamprey. In the PNS, lamprey axons are fully ensheathed either individually or in bundles by cells ortholog to Schwann cells. In the CNS, axons associate with astrocyte orthologs, which contain glycogen and lipid droplets. We suggest that ensheathment, radial sorting, and metabolic support of axons by glial cells predate the evolutionary emergence of myelin in ancient jawed vertebrates.

Contrast enhancement for visualizing neuronal cytoarchitecture by propagation-based x-ray phase-contrast tomography.

Knowledge of the three-dimensional (3d) neuronal cytoarchitecture is an important factor in order to understand the connection between tissue structure and function or to visualize pathological changes in neurodegenerative diseases or tumor development. The gold standard in neuropathology is histology, a technique which provides insights into the cellular organization based on sectioning of the sample. Conventional histology, however, misses the complete 3d information as only individual two-dimensional slices through the object are available. In this work, we use propagation-based phase-contrast x-ray tomography to perform 3d virtual histology on cerebellar tissue from mice. This technique enables us to non-invasively visualize the entire 3d density distribution of the examined samples at isotropic (sub-)cellular resolution. One central challenge, however, of the technique is the fact that contrast for important structural features can be easily lost due to small electron density differences, notably between the cells and surrounding tissue. Here, we evaluate the influence of different embedding media, which are intermediate steps in sample preparation for classical histology, on contrast formation and examine the applicability of the different sample preparations both at a synchrotron-based holotomography setup as well as a laboratory source.

3d Virtual Histology Reveals Pathological Alterations of Cerebellar Granule Cells in Multiple Sclerosis.

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.

Fiber orientation in a whole mouse heart reconstructed by laboratory phase-contrast micro-CT.

Purpose: We present a phase-contrast x-ray tomography study of wild type C57BL/6 mouse hearts as a nondestructive approach to the microanatomy on the scale of the entire excised organ. Based on the partial coherence at a home-built phase-contrast µ - CT setup installed at a liquid metal jet source, we exploit phase retrieval and hence achieve superior image quality for heart tissue, almost comparable to previous synchrotron data on the whole organ scale. Approach: In our work, different embedding methods and heavy metal-based stains have been explored. From the tomographic reconstructions, quantitative structural parameters describing the three-dimensional (3-D) architecture have been derived by two different fiber tracking algorithms. The first algorithm is based on the local gradient of the reconstructed electron density. By performing a principal component analysis on the local structure-tensor of small subvolumes, the dominant direction inside the volume can be determined. In addition to this approach, which is already well established for heart tissue, we have implemented and tested an algorithm that is based on a local 3-D Fourier transform. Results: We showed that the choice of sample preparation influences the 3-D structure of the tissue, not only in terms of contrast but also with respect to the structural preservation. A heart prepared with the evaporation-of-solvent method was used to compare both algorithms. The results of structural orientation were very similar for both approaches. In addition to the determination of the fiber orientation, the degree of filament alignment and local thickness of single muscle fiber bundles were obtained using the Fourier-based approach. Conclusions: Phase-contrast x-ray tomography allows for investigating the structure of heart tissue with an isotropic resolution below 10 µ m . The fact that this is possible with compact laboratory instrumentation opens up new opportunities for screening samples and optimizing sample preparation, also prior to synchrotron beamtimes. Further, results from the structural analysis can help in understanding cardiovascular diseases or can be used to improve computational models of the heart.

Nanoscale x-ray holotomography of human brain tissue with phase retrieval based on multienergy recordings.

X-ray cone-beam holotomography of unstained tissue from the human central nervous system reveals details down to subcellular length scales. This visualization of variations in the electron density of the sample is based on phase-contrast techniques using intensities formed by self-interference of the beam between object and detector. Phase retrieval inverts diffraction and overcomes the phase problem by constraints such as several measurements at different Fresnel numbers for a single projection. Therefore, the object-to-detector distance (defocus) can be varied. However, for cone-beam geometry, changing defocus changes magnification, which can be problematic in view of image processing and resolution. Alternatively, the photon energy can be altered (multi-E). Far from absorption edges, multi-E data yield the wavelength-independent electron density. We present the multi-E holotomography at the Göttingen Instrument for Nano-Imaging with X-Rays (GINIX) setup of the P10 beamline at Deutsches Elektronen-Synchrotron. The instrument is based on a combined optics of elliptical mirrors and an x-ray waveguide positioned in the focal plane for further coherence, spatial filtering, and high numerical aperture. Previous results showed the suitability of this instrument for nanoscale tomography of unstained brain tissue. We demonstrate that upon energy variation, the focal spot is stable enough for imaging. To this end, a double-crystal monochromator and automated alignment routines are required. Three tomograms of human brain tissue were recorded and jointly analyzed using phase retrieval based on the contrast transfer function formalism generalized to multiple photon energies. Variations of the electron density of the sample are successfully reconstructed.

Phase-contrast x-ray tomography of neuronal tissue at laboratory sources with submicron resolution.

Purpose: Recently, progress has been achieved in implementing phase-contrast tomography of soft biological tissues at laboratory sources. This opens up opportunities for three-dimensional (3-D) histology based on x-ray computed tomography ( µ - and nanoCT) in the direct vicinity of hospitals and biomedical research institutions. Combining advanced x-ray generation and detection techniques with phase reconstruction algorithms, 3-D histology can be obtained even of unstained tissue of the central nervous system, as shown, for example, for biopsies and autopsies of human cerebellum. Depending on the setup, i.e., source, detector, and geometric parameters, laboratory-based tomography can be implemented at very different sizes and length scales. We investigate the extent to which 3-D histology of neuronal tissue can exploit the cone-beam geometry at high magnification M using a nanofocus transmission x-ray tube (nanotube) with a 300 nm minimal spot size (Excillum), combined with a single-photon counting camera. Tightly approaching the source spot with the biopsy punch, we achieve high M ≈ 10 1 - 10 2 , high flux density, and exploit the superior efficiency of this detector technology. Approach: Different nanotube configurations such as spot size and flux, M , as well as exposure time, Fresnel number, and coherence are varied and selected in view of resolution, field of view, and/or phase-contrast requirements. Results: The data show that the information content for the cytoarchitecture is enhanced by the phase effect. Comparison of results to those obtained at a microfocus rotating-anode x-ray tomography setup with a high-resolution detector, i.e., in low- M geometry, reveals similar to slightly superior data quality for the nanotube setup. In addition to its compactness, reduced power consumption by a factor of 10 3 , and shorter scan duration, the particular advantage of the nanotube setup also lies in its suitability for pixel detector technology, enabling an increased range of opportunities for applications in laboratory phase-contrast x-ray tomography. Conclusions: The phase retrieval scheme utilized mixes amplitude and phase contrast, with results being robust with respect to reconstruction parameters. Structural information content is comparable to slightly superior to previous results achieved with a microfocus rotating-anode setup but can be obtained in shorter scan time. Beyond advantages as compactness, lowered power consumption, and flexibility, the nanotube setup's scalability in view of the progress in pixel detector technology is particularly beneficial. Further progress is thus likely to bring 3-D virtual histology to the performance in scan time and throughput required for clinical practice in neuropathology.

Multiscale x-ray phase-contrast tomography in a mouse model of transient focal cerebral ischemia.

Cerebral ischemia is associated with a lack of oxygen and high-energy phosphates within the brain tissue, leading to irreversible cell injury. Visualizing these cellular injuries has long been a focus of experimental stroke research with application of immunohistochemistry as one of the standard approaches. It is, however, a destructive imaging technique with non-isotropic resolution, as only the two-dimensional tissue structure of a thin brain section is visualized using optical microscopy and specific stainings. Herein, we extend the structural analysis of mouse brain tissue after cerebral ischemia to the third dimension via microfocus computed tomography (µ-CT). Contrast of the weakly absorbing unstained brain tissue is enhanced by phase contrast. We show that recordings at two different magnifications and fields of view can be combined as a single approach for visualization of the associated structural alterations at isotropic resolution, from the level of the whole organ down to single cells.

Formation and development of the male copulatory organ in the spider Parasteatoda tepidariorum involves a metamorphosis-like process.

Spiders have evolved a unique male copulatory organ, the pedipalp bulb. The morphology of the bulb is species specific and plays an important role in species recognition and prezygotic reproductive isolation. Despite its importance for spider biodiversity, the mechanisms that control bulb development are virtually unknown. We have used confocal laser scanning microscopy (CLSM) and diffusible iodine-based contrast-enhanced micro computed tomography (dice-µCT) to study bulb development in the spider Parasteatoda tepidariorum. These imaging technologies enabled us to study bulb development in situ, without the use of destructive procedures for the first time. We show here that the inflated pedipalp tip in the subadult stage is filled with haemolymph that rapidly coagulates. Coagulation indicates histolytic processes that disintegrate tibia and tarsus, similar to histolytic processes during metamorphosis in holometabolous insects. The coagulated material contains cell inclusions that likely represent the cell source for the re-establishment of tarsus and tibia after histolysis, comparable to the histoblasts in insect metamorphosis. The shape of the coagulated mass prefigures the shape of the adult tarsus (cymbium) like a blueprint for the histoblasts. This suggests a unique role for controlled coagulation after histolysis in the metamorphosis-like morphogenesis of the male pedipalp.

X-ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation.

Imaging techniques are a cornerstone of contemporary biology. Over the last decades, advances in microscale imaging techniques have allowed fascinating new insights into cell and tissue morphology and internal anatomy of organisms across kingdoms. However, most studies so far provided snapshots of given reference taxa, describing organs and tissues under "idealized" conditions. Surprisingly, there is an almost complete lack of studies investigating how an organism's internal morphology changes in response to environmental drivers. Consequently, ecology as a scientific discipline has so far almost neglected the possibilities arising from modern microscale imaging techniques. Here, we provide an overview of recent developments of X-ray computed tomography as an affordable, simple method of high spatial resolution, allowing insights into three-dimensional anatomy both in vivo and ex vivo. We review ecological studies using this technique to investigate the three-dimensional internal structure of organisms. In addition, we provide practical comparisons between different preparation techniques for maximum contrast and tissue differentiation. In particular, we consider the novel modality of phase contrast by self-interference of the X-ray wave behind an object (i.e., phase contrast by free space propagation). Using the cricket Acheta domesticus (L.) as model organism, we found that the combination of FAE fixative and iodine staining provided the best results across different tissues. The drying technique also affected contrast and prevented artifacts in specific cases. Overall, we found that for the interests of ecological studies, X-ray computed tomography is useful when the tissue or structure of interest has sufficient contrast that allows for an automatic or semiautomatic segmentation. In particular, we show that reconstruction schemes which exploit phase contrast can yield enhanced image quality. Combined with suitable specimen preparation and automated analysis, X-ray CT can therefore become a promising quantitative 3D imaging technique to study organisms' responses to environmental drivers, in both ecology and evolution.

Propagation-based phase-contrast x-ray tomography of cochlea using a compact synchrotron source.

We demonstrate that phase retrieval and tomographic imaging at the organ level of small animals can be advantageously carried out using the monochromatic radiation emitted by a compact x-ray light source, without further optical elements apart from source and detector. This approach allows to carry out microtomography experiments which - due to the large performance gap with respect to conventional laboratory instruments - so far were usually limited to synchrotron sources. We demonstrate the potential by mapping the functional soft tissue within the guinea pig and marmoset cochlea, including in the latter case an electrical cochlear implant. We show how 3d microanatomical studies without dissection or microscopic imaging can enhance future research on cochlear implants.

Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography.

Studies of brain cytoarchitecture in mammals are routinely performed by serial sectioning of the specimen and staining of the sections. The procedure is labor-intensive and the 3D architecture can only be determined after aligning individual 2D sections, leading to a reconstructed volume with non-isotropic resolution. Propagation-based x-ray phase-contrast tomography offers a unique potential for high-resolution 3D imaging of intact biological specimen due to the high penetration depth and potential resolution. We here show that even compact laboratory CT at an optimized liquid-metal jet microfocus source combined with suitable phase-retrieval algorithms and a novel tissue preparation can provide cellular and subcellular resolution in millimeter sized samples of mouse brain. We removed water and lipids from entire mouse brains and measured the remaining dry tissue matrix in air, lowering absorption but increasing phase contrast. We present single-cell resolution images of mouse brain cytoarchitecture and show that axons can be revealed in myelinated fiber bundles. In contrast to optical 3D techniques our approach does neither require staining of cells nor tissue clearing, procedures that are increasingly difficult to apply with increasing sample and brain sizes. The approach thus opens a novel route for high-resolution high-throughput studies of brain architecture in mammals.

Imaging of neuronal tissues by x-ray diffraction and x-ray fluorescence microscopy: evaluation of contrast and biomarkers for neurodegenerative diseases.

We have used scanning X-ray diffraction (XRD) and X-ray fluorescence (XRF) with micro-focused synchrotron radiation to study histological sections from human substantia nigra (SN). Both XRF and XRD mappings visualize tissue properties, which are inaccessible by conventional microscopy and histology. We propose to use these advanced tools to characterize neuronal tissue in neurodegeneration, in particular in Parkinson's disease (PD). To this end, we take advantage of the recent experimental progress in x-ray focusing, detection, and use automated data analysis scripts to enable quantitative analysis of large field of views. XRD signals are recorded and analyzed both in the regime of small-angle (SAXS) and wide-angle x-ray scattering (WAXS). The SAXS signal was analyzed in view of the local myelin structure, while WAXS was used to identify crystalline deposits. PD tissue scans exhibited increased amounts of crystallized cholesterol. The XRF analysis showed increased amounts of iron and decreased amounts of copper in the PD tissue compared to the control.