Christos Bikis

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.

Single and double grating-based X-ray microtomography using synchrotron radiation

Hard X-ray phase contrast imaging techniques have become most suitable for the non-destructive three-dimensional visualization of soft tissues at the microscopic level. Among the hard X-ray grating interferometry methods, a single-grating approach (XSGI) has been implemented by simplifying the established double-grating interferometer (XDGI). We quantitatively compare the XSGI and XDGI tomograms of a human nerve and demonstrate that both techniques provide sufficient contrast to allow for the distinction of tissue types. The two-fold binned data show spatial resolution of (5.2 ± 0.6) μm and (10.7 ± 0.6) μm, respectively, underlying the performance of XSGI in soft tissue imaging.

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.

Non-destructive phase contrast hard x-ray imaging to reveal the three-dimensional microstructure of soft and hard tissues

X-ray imaging in the absorption contrast mode is an established method of visualising calcified tissues such as bone and teeth. Physically soft tissues such as brain or muscle are often imaged using magnetic resonance imaging (MRI). However, the spatial resolution of MRI is insufficient for identifying individual biological cells within three-dimensional tissue. X-ray grating interferometry (XGI) has advantages for the investigation of soft tissues or the simultaneous three-dimensional visualisation of soft and hard tissues. Since laboratory microtomography (μCT) systems have better accessibility than tomography set-ups at synchrotron radiation facilities, a great deal of effort has been invested in optimising XGI set-ups for conventional μCT systems. In this conference proceeding, we present how a two-grating interferometer is incorporated into a commercially available nanotom m (GE Sensing and Inspection Technologies GmbH) μCT system to extend its capabilities toward phase contrast. We intend to demonstrate superior contrast in spiders (Hogna radiata (Fam. Lycosidae) and Xysticus erraticus (Fam. Thomisidae)), as well as the simultaneous visualisation of hard and soft tissues. XGI is an imaging modality that provides quantitative data, and visualisation is an important part of biomimetics; consequently, hard X-ray imaging provides a sound basis for bioinspiration, bioreplication and biomimetics and allows for the quantitative comparison of biofabricated products with their natural counterparts.

Automatic histology registration in application to x-ray modalities

Registration of microscope images to Computed Tomography (CT) 3D volumes is a challenging task because it requires not only multi-modal similarity measure but also 2D-3D or slice-to-volume correspondence. This type of registration is usually done manually which is very time-consuming and prone to errors. Recently we have developed the first automatic approach to localize histological sections in μCT data of a jaw bone. The median distance between the automatically found slices and the ground truth was below 35 μm. Here we explore the limitations of the method by applying it to three tomography datasets acquired with grating interferometry, laboratory-based μCT and single-distance phase retrieval. Moreover, we compare the performance of three feature detectors in the proposed framework, i.e. Speeded Up Robust Features (SURF), Scale Invariant Feature Transform (SIFT) and Affine SIFT (ASIFT). Our results show that all the feature detectors performed significantly better on the grating interferometry dataset than on other modalities. The median accuracy for the vertical position was 0.06 mm. Across the feature detector types the smallest error was achieved by the SURF-based feature detector (0.29 mm). Furthermore, the SURF-based method was computationally the most efficient. Thus, we recommend to use the SURF feature detector for the proposed framework.

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.

Automatic deformable registration of histological slides to μCT volume data: DEFORMABLE REGISTRATION FROM HISTOLOGY TO VOLUME DATA

Localizing a histological section in the three‐dimensional dataset of a different imaging modality is a challenging 2D‐3D registration problem. In the literature, several approaches have been proposed to solve this problem; however, they cannot be considered as fully automatic. Recently, we developed an automatic algorithm that could successfully find the position of a histological section in a micro computed tomography (μCT) volume. For the majority of the datasets, the result of localization corresponded to the manual results. However, for some datasets, the matching μCT slice was off the ground‐truth position. Furthermore, elastic distortions, due to histological preparation, could not be accounted for in this framework.

A quantitative correction for phase wrapping artifacts in hard X-ray grating interferometry

X-ray grating interferometry-based computed tomography is a phase contrast imaging technique that provides non-destructive, quantitative, and three-dimensional visualization with contrast superior to traditional absorption-based techniques, especially for materials primarily composed of low Z elements, such as biological tissues. However, it relies on measurements of the lateral shift of an interference pattern and is thus susceptible to so-called phase wrapping artifacts, which mainly occur at the sample-air interface. In this work, we present an algorithm for removal of such artifacts in the case of cylindrical samples and an experiment to verify its accuracy. The proposed algorithm is applied to the sinogram after phase retrieval and prior to reconstruction by finding sample edges with the absorption sinogram and replacing regions of the phase wrapped sinogram with modeled data. Our measurements show that the algorithm removes artifacts and produces more accurate δ values, as validated by measurements without phase wrapping. Our correction algorithm allows for measurements without submerging the sample in a water bath, simplifying the experimental setup and avoiding motion artifacts from gas bubbles.X-ray grating interferometry-based computed tomography is a phase contrast imaging technique that provides non-destructive, quantitative, and three-dimensional visualization with contrast superior to traditional absorption-based techniques, especially for materials primarily composed of low Z elements, such as biological tissues. However, it relies on measurements of the lateral shift of an interference pattern and is thus susceptible to so-called phase wrapping artifacts, which mainly occur at the sample-air interface. In this work, we present an algorithm for removal of such artifacts in the case of cylindrical samples and an experiment to verify its accuracy. The proposed algorithm is applied to the sinogram after phase retrieval and prior to reconstruction by finding sample edges with the absorption sinogram and replacing regions of the phase wrapped sinogram with modeled data. Our measurements show that the algorithm removes artifacts and produces more accurate δ values, as validated by measurements ...

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.