3D X-ray Histology for the Investigation of Temporal Lobe Epilepsy in a Mouse Model.

The most common form of epilepsy among adults is mesial temporal lobe epilepsy (mTLE), with seizures often originating in the hippocampus due to abnormal electrical activity. The gold standard for the histopathological analysis of mTLE is histology, which is a two-dimensional technique. To fill this gap, we propose complementary three-dimensional (3D) X-ray histology. Herein, we used synchrotron radiation-based phase-contrast microtomography with 1.6 µm-wide voxels for the post mortem visualization of tissue microstructure in an intrahippocampal-kainate mouse model for mTLE. We demonstrated that the 3D X-ray histology of unstained, unsectioned, paraffin-embedded brain hemispheres can identify hippocampal sclerosis through the loss of pyramidal neurons in the first and third regions of the Cornu ammonis as well as granule cell dispersion within the dentate gyrus. Morphology and density changes during epileptogenesis were quantified by segmentations from a deep convolutional neural network. Compared to control mice, the total dentate gyrus volume doubled and the granular layer volume quadrupled 21 days after injecting kainate. Subsequent sectioning of the same mouse brains allowed for benchmarking 3D X-ray histology against well-established histochemical and immunofluorescence stainings. Thus, 3D X-ray histology is a complementary neuroimaging tool to unlock the third dimension for the cellular-resolution histopathological analysis of mTLE.

Implementation of a double-grating interferometer for phase-contrast computed tomography in a conventional system nanotom® m.

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.

Three-dimensional imaging and analysis of entire peripheral nerves after repair and reconstruction.

BACKGROUND: We wanted to achieve a three-dimensional (3D), non-destructive imaging and automatic post-analysis and evaluation of reconstructed peripheral nerves without involving cutting and staining processes. NEW METHOD: We used a laboratory-based micro computed tomography system for imaging, as well as a custom analysis protocol. The sample preparation was also adapted in order to achieve 3D images with true micrometer resolution and suitable contrast. RESULTS: Analysis of the acquired tomograms enabled the quantitative assessment of 3D tissue structures, i.e., surface morphology, nerve fascicles, nerve tissue volume, geometry, and vascular regrowth. The resulting data showed significant differences between operated animals and non-operated controls. COMPARISON WITH EXISTING METHODS: Our approach avoids the sampling error associated with conventional 2D visualization approaches and holds promise for automation of the analysis of large series of datasets. CONCLUSIONS: We have presented a potential way for 3D imaging and analysis of entire regenerated nerves non-destructively, paving the way for high-throughput analysis of therapeutic conditions of treating adult nerve injuries.

Three-dimensional and non-destructive characterization of nerves inside conduits using laboratory-based micro computed tomography.

BACKGROUND: Histological assessment of peripheral nerve regeneration in animals is tedious, time-consuming and challenging for three-dimensional analysis. NEW METHOD: The present study reports on how and to what extent micro computed tomography of paraffin-embedded samples can provide a reliable three-dimensional approach for quantitative analysis of peripheral nerves. RESULTS: Rat sciatic nerves were harvested, formalin-fixated, positioned into nerve conduits (NC), paraffin-embedded, and imaged using a laboratory-based X-ray microtomography system with an isotropic voxel length of 4µm. Suitable quantitative measures were identified and automatically evaluated, i.e. nerve length, cross-sectional area and volume, as well as vascular structures, to be used as an assessment and comparison indicator of regeneration quality. COMPARISON WITH EXISTING METHODS: Compared to imaging using contrast agents, the investigated specimens can subsequently undergo the conventional histological analysis without requiring additional preparation steps. Contrast and spatial resolution are also increased significantly. CONCLUSIONS: We demonstrate the potential of the micro computed tomography for non-destructive monitoring of peripheral nerves inside the conduits.

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

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.

Mineralization of Early Stage Carious Lesions In Vitro-A Quantitative Approach.

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.