Gabriel Schweighauser

Extending two-dimensional histology into the third dimension through conventional micro computed tomography.

Histological examination achieves sub-micrometer resolution laterally. In the third dimension, however, resolution is limited to section thickness. In addition, histological sectioning and mounting sections on glass slides introduce tissue-dependent stress and strain. In contrast, state-of-the-art hard X-ray micro computed tomography (μCT) systems provide isotropic sub-micrometer resolution and avoid sectioning artefacts. The drawback of μCT in the absorption contrast mode for visualising physically soft tissue is a low attenuation difference between anatomical features. In this communication, we demonstrate that formalin-fixed paraffin-embedded human cerebellum yields appropriate absorption contrast in laboratory-based μCT data, comparable to conventional histological sections. Purkinje cells, for example, are readily visible. In order to investigate the pros and cons of complementary approaches, two- and three-dimensional data were manually and automatically registered. The joint histogram of histology and the related μCT slice allows for a detailed discussion on how to integrate two-dimensional information from histology into a three-dimensional tomography dataset. This methodology is not only rewarding for the analysis of the human cerebellum, but it also has relevance for investigations of tissue biopsies and post-mortem applications. Our data indicate that laboratory-based μCT as a modality can fill the gap between synchrotron radiation-based μCT and histology for a variety of tissues. As the information from haematoxylin and eosin (H&E) stained sections and μCT data is related, one can colourise local X-ray absorption values according to the H&E stain. Hence, μCT data can correlate and virtually extend two-dimensional (2D) histology data into the third dimension.

Tomographic brain imaging with nucleolar detail and automatic cell counting

Brain tissue evaluation is essential for gaining in-depth insight into its diseases and disorders. Imaging the human brain in three dimensions has always been a challenge on the cell level. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Herein, we show that hard X-ray phase tomography can visualise a volume of up to 43 mm3 of human post mortem or biopsy brain samples, by demonstrating the method on the cerebellum. We automatically identified 5,000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, we highlight that three-dimensional data allows for the segmentation of sub-cellular structures, including dendritic tree and Purkinje cell nucleoli, without dedicated staining. The method suggests that automatic cell feature quantification of human tissues is feasible in phase tomograms obtained with isotropic resolution in a label-free manner.

Lewy pathology in Parkinson's disease consists of a crowded organellar membranous medley

Parkinson’s disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites, which are neuronal inclusions that are immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of this Lewy pathology is crucial to understanding pathogenesis and progression of the disease. Using correlative light and electron microscopy/tomography on brain tissue from five Parkinson’s disease brain donors to identify α-synuclein immunopositive structures, we show that the majority of Lewy pathology including Lewy bodies and Lewy neurites primarily consists of a crowded membranous medley of vesicular structures and dysmorphic organelles. Only a small fraction of observed Lewy bodies contained predominant proteinaceous filaments, as previously described. The crowding of organellar components was confirmed by STED-based super-resolution microscopy, and high lipid content within the α-synuclein immunopositive inclusions was corroborated by confocal imaging, CARS/FTIR imaging and lipidomics. Applying this correlative high-resolution imaging and biophysical approach, we discovered in the postmortem brain of Parkinson’s patients a subcellular protein-lipid compartmentalization not previously described in Lewy pathology.

Hard X‐Ray Nanoholotomography: Large‐Scale, Label‐Free, 3D Neuroimaging beyond Optical Limit

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.

Three-dimensional imaging of human brain tissues using absorption-contrast high-resolution X-ray tomography

Our body is hierarchically organized down to individual cells. Cutting-edge clinical imaging facilities reach a spatial resolution of a fraction of a millimeter, living cells invisible. A decade ago, post-mortem X-ray imaging by means of synchrotron radiation enabled the identification of Os-stained ganglion and unstained Purkinje cells. Very recently, even sub-cellular structures, such as nucleolus and the dendritic tree of Purkinje cells, were extracted by means of phase-contrast single-distance synchrotron radiation-based hard X-ray tomography. At the same time, conventional absorption-contrast, laboratory-based micro computed tomography was successfully applied to visualize brain components including individual Purkinje cells within a cerebellum specimen. Thus, the goal of isotropic-cellular-resolution visualization of soft tissues within a laboratory environment without application of any dedicated contrast agent was achieved. In this communication, we are discussing (1) to which extend the quality gain of the laboratory-based absorption-contrast tomography can be driven with respect to optical microscopy of stained tissue sections and (2) what value such a technique would add. As a proof of principle, four histological sections were affine-registered to corresponding three-dimensional (3D) tomography dataset. We are discussing a semi-automatic landmark-based 2D-3D registration framework and compare registration results based on mean square difference (MSD) metrics.

Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers (Stratum moleculare and Stratum granulosum), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H&E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology.

Imaging cellular and subcellular structure of human brain tissue using micro computed tomography

Brain tissues have been an attractive subject for investigations in neuropathology, neuroscience, and neurobiol- ogy. Nevertheless, existing imaging methodologies have intrinsic limitations in three-dimensional (3D) label-free visualisation of extended tissue samples down to (sub)cellular level. For a long time, these morphological features were visualised by electron or light microscopies. In addition to being time-consuming, microscopic investigation includes specimen fixation, embedding, sectioning, staining, and imaging with the associated artefacts. More- over, optical microscopy remains hampered by a fundamental limit in the spatial resolution that is imposed by the diffraction of visible light wavefront. In contrast, various tomography approaches do not require a complex specimen preparation and can now reach a true (sub)cellular resolution. Even laboratory-based micro computed tomography in the absorption-contrast mode of formalin-fixed paraffin-embedded (FFPE) human cerebellum yields an image contrast comparable to conventional histological sections. Data of a superior image quality was obtained by means of synchrotron radiation-based single-distance X-ray phase-contrast tomography enabling the visualisation of non-stained Purkinje cells down to the subcellular level and automated cell counting. The question arises, whether the data quality of the hard X-ray tomography can be superior to optical microscopy. Herein, we discuss the label-free investigation of the human brain ultramorphology be means of synchrotron radiation-based hard X-ray magnified phase-contrast in-line tomography at the nano-imaging beamline ID16A (ESRF, Grenoble, France). As an example, we present images of FFPE human cerebellum block. Hard X-ray tomography can provide detailed information on human tissues in health and disease with a spatial resolution below the optical limit, improving understanding of the neuro-degenerative diseases.

X-ray micro-tomography for investigations of brain tissues on cellular level

X-ray imaging in absorption contrast mode is well established for hard tissue visualization. However, performance for lower density materials is limited due to a reduced contrast. Our aim is three-dimensional (3D) characterization of micro-morphology of human brain tissues down to (sub-)cellular resolution within a laboratory environment. Using the laboratory-based microtomography (μCT) system nanotom m (GE Sensing and Inspection Technologies GmbH, Wunstorf, Germany) and synchrotron radiation at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK), we have acquired 3D data with a resolution down to 0.45 μm for visualization of a human cerebellum specimen down to cellular level. We have shown that all selected modalities, namely laboratory-based absorption contrast micro-tomography (LBμCT), synchrotron radiation based in-line single distance phase contrast tomography (SDPR) and synchrotron radiation based single-grating interferometry (GI), can reach cellular resolution for tissue samples with a size in the mm-range. The results are discussed qualitatively in comparison to optical microscopy of haematoxylin and eosin (HE) stained sections. As phase contrast yields to a better data quality for soft tissues and in order to overcome restrictions of limited beamline access for phase contrast measurements, we have equipped the μCT system nanotom m with a double-grating phase contrast set-up. Preliminary experimental results of a knee sample consisting of a bony part and a cartilage demonstrate that phase contrast data exhibits better quality compared to absorption contrast. Currently, the set-up is under adjustment. It is expected that cellular resolution would also be achieved. The questions arise (1) what would be the quality gain of laboratory-based phase contrast in comparison to laboratory-based absorption contrast tomography and (2) could laboratory-based phase contrast data provide comparable results to synchrotron radiation based phase contrast data.

High-resolution synchrotron radiation-based phase tomography of the healthy and epileptic brain

Phase-contrast micro-tomography using synchrotron radiation has yielded superior soft tissue visualization down to the sub-cellular level. The isotropic spatial resolution down to about one micron is comparable to the one of histology. The methods, however, provide different physical quantities and are thus complementary, also allowing for the extension of histology into the third dimension. To prepare for cross-sectional animal studies on epilepsy, we have standardized the specimen’s preparation and scanning procedure for mouse brains, so that subsequent histology remains entirely unaffected and scanning of all samples (n = 28) is possible in a realistic time frame. For that, we have scanned five healthy and epileptic mouse brains at the ID19 beamline, ESRF, Grenoble, France, using grating- and propagation-based phase contrast micro-tomography. The resulting datasets clearly show the cortex, ventricular system, thalamus, hypothalamus, and hippocampus. Our focus is on the latter, having planned kainate-induced epilepsy experiments. The cell density and organization in the dentate gyrus and Ammon’s horn region were clearly visualized in control animals. This proof of principle was required to initiate experiment. The resulting three-dimensional data have been correlated to histology. The goal is a brain-wide quantification of cell death or structural reorganization associated with epilepsy as opposed to histology alone that represents small volumes of the total brain only. Thus, the proposed technique bears the potential to correlate the gold standard in analysis with independently obtained data sets. Such an achievement also fuels interest for other groups in neuroscience research to closely collaborate with experts in phase micro-tomography.

Computational cell quantification in the human brain tissues based on hard x-ray phase-contrast tomograms

Cell visualization and counting plays a crucial role in biological and medical research including the study of neurodegenerative diseases. The neuronal cell loss is typically determined to measure the extent of the disease. Its characterization is challenging because the cell density and size already differs by more than three orders of magnitude in a healthy cerebellum. Cell visualization is commonly performed by histology and fluorescence microscopy. These techniques are limited to resolve complex microstructures in the third dimension. Phase- contrast tomography has been proven to provide sufficient contrast in the three-dimensional imaging of soft tissue down to the cell level and, therefore, offers the basis for the three-dimensional segmentation. Within this context, a human cerebellum sample was embedded in paraffin and measured in local phase-contrast mode at the beamline ID19 (ESRF, Grenoble, France) and the Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK). After the application of Frangi-based filtering the data showed sufficient contrast to automatically identify the Purkinje cells and to quantify their density to 177 cells per mm3 within the volume of interest. Moreover, brain layers were segmented in a region of interest based on edge detection. Subsequently performed histological analysis validated the presence of the cells, which required a mapping from the two- dimensional histological slices to the three-dimensional tomogram. The methodology can also be applied to further tissue types and shows potential for the computational tissue analysis in health and disease.