Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

Depth-dependent anisotropies of amides and sugar in perpendicular and parallel sections of articular cartilage by Fourier transform infrared imaging

Full thickness blocks of canine humeral cartilage were microtomed into both perpendicular sections and a series of 100 parallel sections, each 6 μm thick. Fourier transform infrared (IR) imaging was used to image each tissue section eleven times under different IR polarizations (from 0° to 180° polarization states in 20° increments and with an additional 90° polarization), at a spatial resolution of 6.25 μm and a wavenumber step of 8 cm^?1. With increasing depth from the articular surface, amide anisotropies increased in the perpendicular sections and decreased in the parallel sections. Both types of tissue sectioning identified a 90° difference between amide I and amide II in the superficial zone (SZ) of cartilage. The fibrillar distribution in the parallel sections from the SZ was shown to not be random. Sugar had a weak but recognizable anisotropy in the upper part of the radial zone (RZ) in the perpendicular sections. The depth‐dependent anisotropic data were fitted with a theoretical equation that contained three signature parameters, which illustrate the arcade structure of collagens with the aid of a fibril model. Fourier‐transform IR imaging of both perpendicular and parallel sections provides the possibility of determining the three‐dimensional macromolecular structures in articular cartilage. Being sensitive to the orientation of the macromolecular structure in healthy articular cartilage aids the prospect of detecting the early onset of the tissue degradation that may lead to pathological conditions such as osteoarthritis. Microsc. Res. Tech. 74:122–132, 2011. © 2010 Wiley‐Liss, Inc.     

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.     

Quantitative zonal differentiation of articular cartilage by microscopic magnetic resonance imaging,polarized light microscopy,and Fourier‐transform infrared imaging

This study aimed to synchronize the zonal differentiation of the full‐thickness articular cartilage by three micro‐imaging techniques, namely microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and Fourier‐transform infrared imaging (FTIRI). Eighteen cartilage‐bone blocks from three canine humeral joints were imaged by: (a) µMRI T₂ relaxation at 0° and 55° orientations in a 7 T magnetic field, (b) PLM optical retardation and azimuthal angle, and (c) FTIRI amide I and amide II anisotropies at 0° and 90° polarizations relative to the articular surface. In addition, µMRI T₁ relaxation was imaged before and after the tissue being immersed in gadolinium (contrast agent) solution, to calculate the proteoglycan concentration. A set of previously established criteria in cartilage imaging was revised. The new criteria could simultaneously correlate the thicknesses of the three consecutive subtissue zones in articular cartilage among these imaging techniques. Microsc. Res. Tech. 76:625–632, 2013. © 2013 Wiley Periodicals, Inc.     

Virtual histology by means of high‐resolution X‐ray CT

Micro‐CT is a non‐destructive technique for 3D tomographic investigation of an object. A 3D representation of the internal structure is calculated based on a series of X‐ray radiographs taken from different angles. The spatial resolution of current laboratory‐used micro‐CT systems has come down over the last years from a few tens of microns to a few microns. This opens the possibility to perform histological investigations in 3D on a virtual representation of a sample, referred to as virtual 3D histology. The advantage of micro‐CT based virtual histology is the immediate and automated 3D visualization of the sample without prior slicing, sample preparation like decalcification, photographing and aligning. This not only permits a drastic reduction in preparation time but also offers the possibility to easily investigate objects that are difficult to slice. This article presents results that were obtained on punch biopsies of horse skin, (dental) alveolus of ponies and chondro‐osseous samples from the tarsus of foals studied with the new high resolution micro‐CT set‐up (HRXCT) at the Ghent University (Belgium) ( http://www.ugct.ugent.be ). This state‐of‐the‐art set‐up provides a 1 micron resolution and is therefore ideally suited for a direct comparison with standard light microscopy–based histology.     

Micro‐CT versus synchrotron radiation phase contrast imaging of human cochlea

High‐resolution images of the cochlea are used to develop atlases to extract anatomical features from low‐resolution clinical computed tomography (CT) images. We compare visualization and contrast of conventional absorption‐based micro‐CT to synchrotron radiation phase contrast imaging (SR‐PCI) images of whole unstained, nondecalcified human cochleae. Three cadaveric cochleae were imaged using SR‐PCI and micro‐CT. Images were visually compared and contrast‐to‐noise ratios (CNRs) were computed from n = 27 regions‐of‐interest (enclosing soft tissue) for quantitative comparisons. Three‐dimensional (3D) models of cochlear internal structures were constructed from SR‐PCI images using a semiautomatic segmentation method. SR‐PCI images provided superior visualization of soft tissue microstructures over conventional micro‐CT images. CNR improved from 7.5 ± 2.5 in micro‐CT images to 18.0 ± 4.3 in SR‐PCI images (p < 0.0001). The semiautomatic segmentations yielded accurate reconstructions of 3D models of the intracochlear anatomy. The improved visualization, contrast and modelling achieved using SR‐PCI images are very promising for developing atlas‐based segmentation methods for postoperative evaluation of cochlear implant surgery.     

Automatic deformable registration of histological slides to μCT 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. In the current study, we introduce two optimization frameworks based on normalized mutual information, which enabled us to accurately register histology slides to volume data. The rigid approach allocated 81 % of histological sections with a median position error of 8.4 μm in jaw bone datasets, and the deformable approach improved registration by 33 μm with respect to the median distance error for four histological slides in the cerebellum dataset.     

Application of design‐based stereology for estimation of absolute volume and surface area of the articular and calcified cartilage compartments of undecalcified human femoral heads

Design‐based stereological methods using systematic uniform random sampling, the Cavalieri estimator and vertical sections are used to investigate undecalcified human femoral heads. Ten entire human femoral heads, obtained from normal women and normal men, were systematically sampled and thin undecalcified vertical sections were obtained. Absolute volumes and surface areas of the entire femoral head, the articular cartilage and the calcified cartilage compartments were estimated. In addition, the average thickness of the articular cartilage and the calcified cartilage were calculated. The stereological procedures applied to the human femoral heads resulted in average coefficient of errors, which were 0.03–0.06 for the volume estimates and 0.03–0.04 for the surface area estimates. We conclude that design‐based stereology using the Cavalieri estimator and vertical sections can successfully be used in large undecalcified tissue specimens, like the human femoral head, to estimate the absolute volume and surface area of macroscopic as well as of microscopic tissue compartments. The application of well‐known design‐based stereological methods carries potential advantage for investigating the pathology in inflammatory and degenerative joint diseases.     

Three‐dimensional visualization of rat retina by X‐ray differential phase contrast tomographic microscopy

The retina is one of the most tiny and sophisticated tissues of the body. Three dimensional (3D) visualization of the whole retina is valuable both in clinical and research arenas. The tissue has been predominantly assessed by time‐consuming histopathology and optical coherence tomography (OCT) in research and clinical arenas. However, none of the two methods can provide 3D imaging of the retina. The purpose of this study is to give a volumetric visualization of rat retina at submicron resolution, using an emerging imaging technique‐phase‐contrast X‐ray CT. A Sprague‐Dawley (SD) rat eye specimen was scanned with X‐ray differential phase contrast tomographic microscopy (DPC‐microCT) equipped at the Swiss Light Source synchrotron. After scanning, the specimen was subjected to routine histology procedures and severed as a reference. The morphological characteristics and signal features of the retina in the DPC‐microCT images were evaluated. The total retina and its sublayers thicknesses were measured on the DPC‐microCT images and compared with those obtained from the histological sections. The retina structures revealed by DPC‐microCT were highly consistent with the histological section. In this study, we achieved nondestructive 3D visualization of SD rat retina. In addition to detailed anatomical structures, the objective parameters provided by DPC‐microCT make it a useful tool for retinal research and disease diagnosis in the early stage.     

Vascular imaging with contrast agent in hard and soft tissues using microcomputed‐tomography

Vascularization is essential for many tissues and is a main requisite for various tissue‐engineering strategies. Different techniques are used for highlighting vasculature, in vivo and ex vivo, in 2‐D or 3‐D including histological staining, immunohistochemistry, radiography, angiography, microscopy, computed tomography (CT) or micro‐CT, both stand‐alone and synchrotron system. Vascularization can be studied with or without a contrast agent. This paper presents the results obtained with the latest Skyscan micro‐CT (Skyscan 1272, Bruker, Belgium) following barium sulphate injection replacing the bloodstream in comparison with results obtained with a Skyscan In Vivo 1076. Different hard and soft tissues were perfused with contrast agent and were harvested. Samples were analysed using both forms of micro‐CT, and improved results were shown using this new micro‐CT. This study highlights the vasculature using micro‐CT methods. The results obtained with the Skyscan 1272 are clearly defined compared to results obtained with Skyscan 1076. In particular, this instrument highlights the high number of small vessels, which were not seen before at lower resolution. This new micro‐CT opens broader possibilities in detection and characterization of the 3‐D vascular tree to assess vascular tissue engineering strategies.     

Feasibility of detecting trabecular bone around percutaneous titanium implants in rabbits by in vivo microfocus computed tomography

Objectives: The goal of this study was to examine the feasibility of in vivo imaging of trabecular bone around titanium implants by means of microfocus computed tomography (micro‐CT) and the use of rabbits for this purpose. Materials and Methods: Ten male rabbits type Hollander, received a titanium implant (1.7 mm diameter and 10 mm length) in the trabecular bone of the left tibia. Seven weeks later a micro‐CT scan was taken. Four rabbits were used to monitor potential harmful effects from X‐ray absorption until 4 weeks after scanning. A second group of six rabbits was used for testing the hypothesis that a good correlation exists between in vivo micro‐CT images and histological images of trabecular bone around titanium implants. The six rabbits were scanned and sacrificed immediately. The tibias were extracted and submitted to standard histological procedures. This resulted in a total of 12 histological sections and their corresponding 12 micro‐CT images. Bone area measurements were performed at the left and right side of the implant in three regions: 0–500, 500–1000 and 1000–1500 μm distance from the implant interface. Intra‐class correlations (ICC) were calculated between both techniques. Results: The four rabbits did not show any sign of radiodermatitis 4 weeks after scanning. In the micro‐CT images of the group of six rabbits, trabeculae are visible, but not well defined, due to the presence of noise in the image. The ICC for the right implant side were 0.44 for zone 0–500 μm, 0.48 for zone 500–1000 μm and 0.40 for zone 1000–1500 μm. The ICC for the left implant side could not be calculated. Conclusion: A low agreement was found between the bone measurements from histology and in vivo micro‐CT images. The use of the in vivo micro‐CT for trabecular bone imaging around metallic implants should be restricted to track tendencies in follow‐up studies.     

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.     

3D reconstruction of histological sections: Application to mammary gland tissue

In this article, we present a novel method for the automatic 3D reconstruction of thick tissue blocks from 2D histological sections. The algorithm completes a high‐content (multiscale, multifeature) imaging system for simultaneous morphological and molecular analysis of thick tissue samples. This computer‐based system integrates image acquisition, annotation, registration, and three‐dimensional reconstruction. We present an experimental validation of this tool using both synthetic and real data. In particular, we present the 3D reconstruction of an entire mouse mammary gland and demonstrate the integration of high‐resolution molecular data. Microsc. Res. Tech. 73:1019–1029, 2010. © 2010 Wiley‐Liss, Inc.     

Techniques for assessment of wear between human cartilage surfaces

Osteoarthritis (OA) is a disease of joints, affecting a large number of people worldwide. One of the symptoms of OA is wear of articular cartilage; it is thought that among other factors this may be due to failure of lubrication. Injection of bio-lubricants into a joint may remedy this problem. Wear of cartilage and its prevention is a focus of much interest. The present paper describes wear tests performed using human cartilage on cartilage under various working conditions. Several techniques assessing wear are described, such as changes in surface morphology using optical profilometry and variation in the content of collagen and proteoglycans (PG) in the lubricating solution. Of all these techniques the PG content analysis was found to be the most efficient one.     

The interface region between articular cartilage and bone by μMRI and PLM at microscopic resolutions

This dual-modality microscopic imaging study quantifies the interface region between the noncalcified cartilage and the subchondral bone plate, which includes the deep portion of the noncalcified articular cartilage and the zone of calcified cartilage (ZCC). This interface region is typically not visible in routine MRI but becomes visible in MRI with the application of an ultra-short echo time (UTE) sequence. A number of cartilage-bone blocks from a well-documented canine humeral head were harvested for imaging by microscopic MRI (μMRI) and PLM (polarized light microscopy). In μMRI, T2 anisotropic images were acquired by 2D gradient-echo, magnetization-prepared spin-echo and UTE sequences at the 0° and 55° (the magic angle) orientations at 11.7 μm/pixel resolution. In PLM, quantitative optical retardation (nm) and collagen orientation (°) were mapped from the thin sections from the same μMRI specimens at 0.5–2 μm pixel resolutions. The orientational and organizational architecture of the collagen matrix in this interface region was quantified and correlated between the complementary imaging. The magic angle effect as seen in the noncalcified cartilage was statistically confirmed in ZCC in μMRI, which was further supported by quantitative PLM. With an enhanced understanding of the tissue properties in this important interface region, it will potentially be possible to monitor the changes of this tissue region which is instrumental to the initiation and development of osteoarthritis and other joint diseases.     

Framework for morphometric classification of cells in imaging flow cytometry

Imaging flow cytometry is an emerging technology that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy. It allows high‐throughput imaging of cells with good spatial resolution, while they are in flow. This paper proposes a general framework for the processing/classification of cells imaged using imaging flow cytometer. Each cell is localized by finding an accurate cell contour. Then, features reflecting cell size, circularity and complexity are extracted for the classification using SVM. Unlike the conventional iterative, semi‐automatic segmentation algorithms such as active contour, we propose a noniterative, fully automatic graph‐based cell localization. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label‐free leukaemia cell‐lines MOLT, K562 and HL60 from video streams captured using custom fabricated cost‐effective microfluidics‐based imaging flow cytometer. The proposed system is a significant development in the direction of building a cost‐effective cell analysis platform that would facilitate affordable mass screening camps looking cellular morphology for disease diagnosis.     

Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

Alteration of cartilage matrix morphology with histological processing

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neontal rats was seen by light microscopy to: (1) consist of elements, 0·5 μm thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.     

Investigation of inner ear anatomy in mouse using X‐ray phase contrast tomography

A thorough understanding of inner ear anatomy is important for investigators. However, investigation of the mouse inner ear is difficult due to the limitations of imaging techniques. X‐ray phase contrast tomography increases contrast 100–1,000 times compared with conventional X‐ray imaging. This study aimed to investigate inner ear anatomy in a fresh post‐mortem mouse using X‐ray phase contrast tomography and to provide a comprehensive atlas of microstructures with less tissue deformation. All experiments were performed in accordance with our institution's guidelines on the care and use of laboratory animals. A fresh mouse cadaver was scanned immediately after sacrifice using an inline phase contrast tomography system. Slice images were reconstructed using a filtered back‐projection (FBP) algorithm. Standardized axial and coronal planes were adjusted with a multi‐planar reconstruction method. Some three‐dimensional (3D) objects were reconstructed by surface rendering. The characteristic features of microstructures, including otoconia masses of the saccular and utricular maculae, superior and inferior macula cribrosae, single canal, modiolus, and osseous spiral lamina, were described in detail. Spatial positions and relationships of the vestibular structures were exhibited in 3D views. This study investigated mouse inner ear anatomy and provided a standardized presentation of microstructures. In particular, otoconia masses were visualized in their natural status without contrast for the first time. The comprehensive anatomy atlas presented in this study provides an excellent reference for morphology studies of the inner ear.