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.     

Comparison of three‐dimensional analysis and stereological techniques for quantifying lithium‐ion battery electrode microstructures

Lithium‐ion battery performance is intrinsically linked to electrode microstructure. Quantitative measurement of key structural parameters of lithium‐ion battery electrode microstructures will enable optimization as well as motivate systematic numerical studies for the improvement of battery performance. With the rapid development of 3‐D imaging techniques, quantitative assessment of 3‐D microstructures from 2‐D image sections by stereological methods appears outmoded; however, in spite of the proliferation of tomographic imaging techniques, it remains significantly easier to obtain two‐dimensional (2‐D) data sets. In this study, stereological prediction and three‐dimensional (3‐D) analysis techniques for quantitative assessment of key geometric parameters for characterizing battery electrode microstructures are examined and compared. Lithium‐ion battery electrodes were imaged using synchrotron‐based X‐ray tomographic microscopy. For each electrode sample investigated, stereological analysis was performed on reconstructed 2‐D image sections generated from tomographic imaging, whereas direct 3‐D analysis was performed on reconstructed image volumes. The analysis showed that geometric parameter estimation using 2‐D image sections is bound to be associated with ambiguity and that volume‐based 3‐D characterization of nonconvex, irregular and interconnected particles can be used to more accurately quantify spatially‐dependent parameters, such as tortuosity and pore‐phase connectivity.     

Stereological estimation of surface area and barrier thickness of fish gills in vertical sections

Previous morphometric methods for estimation of the volume of components, surface area and thickness of the diffusion barrier in fish gills have taken advantage of the highly ordered structure of these organs for sampling and surface area estimations, whereas the thickness of the diffusion barrier has been measured orthogonally on perpendicularly sectioned material at subjectively selected sites. Although intuitively logical, these procedures do not have a demonstrated mathematical basis, do not involve random sampling and measurement techniques, and are not applicable to the gills of all fish. The present stereological methods apply the principles of surface area estimation in vertical uniform random sections to the gills of the Brazilian teleost Arapaima gigas. The tissue was taken from the entire gill apparatus of the right‐hand or left‐hand side (selected at random) of the fish by systematic random sampling and embedded in glycol methacrylate for light microscopy. Arches from the other side were embedded in Epoxy resin. Reference volume was estimated by the Cavalieri method in the same vertical sections that were used for surface density and volume density measurements. The harmonic mean barrier thickness of the water‐blood diffusion barrier was calculated from measurements taken along randomly selected orientation lines that were sine‐weighted relative to the vertical axis. The values thus obtained for the anatomical diffusion factor (surface area divided by barrier thickness) compare favourably with those obtained for other sluggish fish using existing methods.     

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques

Over the last two decades, several different preparative techniques have been developed to investigate frozen‐hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high‐pressure freezing with plunge freezing, and block faces with frozen‐hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high‐pressure freezing proved optimal for high‐resolution studies and provided the best ultrastructural preservation. A combination of these fast‐freezing techniques with cryo‐ultramicrotomy yielded well‐preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo‐sections, and – after evaporating a heavy metal and carbon onto the surface – are stable enough in the electron beam to provide high‐resolution images of large surface areas for statistical analysis in a cryo‐SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen‐hydrated material.     

Structural characteristics of stored black beans (Phaseolus vulgaris L.)

Scanning electron microscopy (SEM) was used to study and compare the structural characteristics of black beans after 2 years of storage under ambient conditions (AC) and refrigerated hypobaric conditions (RHC). The exterior surface of the seed coats of the black beans were covered with small flake-like materials randomly clustered together. The hilar regions of black beans stored under either AC or RHC were similar, and the “linea lucida” exhibited well defined double palisade cell layers in both bean treatments. Cross sections of the seed coats revealed that the parenchymal cell layers of the beans stored under AC were disrupted and frequently exhibited large intercellular spaces between the cell layers. The parenchymal cell layers of the beans stored under RHC demonstrated little disruption of the cell layers, with only occasional intercellular spaces between the cell layers. Cotyledon cross sections of blackbeans showed that the cotyledon cells of the beans stored under RHC exhibited many large intercellular spaces characteristic of normal beans. Conversely, cotyledon cross sections of the beans stored under AC exhibited cotyledon cells with few small intercellular spaces characteristic of hard-to-cook beans.     

Tissue shrinkage and unbiased stereological estimation of particle number and size*

This paper is a review of the stereological problems related to the unbiased estimation of particle number and size when tissue deformation is present. The deformation may occur during the histological processing of the tissue. It is especially noted that the widely used optical disector may be biased by dimensional changes in the z‐axis, i.e. the direction perpendicular to the section plane. This is often the case when frozen sections or vibratome sections are used for the stereological measurements. The present paper introduces new estimators to be used in optical fractionator and optical disector designs; the first is, as usual, the simplest and most robust. Finally, it is stated that when tissue deformation only occurs in the z‐direction, unbiased estimation of particle size with several estimators is possible.     

Three‐dimensional digital reconstruction of skin epidermis and dermis

This study describes how three‐dimensional (3D) human skin tissue is reconstructed, and provides digital anatomical data for the physiological structure of human skin tissue based on large‐scale thin serial sections. Human skin samples embedded in paraffin were cut serially into thin sections and then stained with hematoxylin‐eosin. Images of serial sections obtained from lighting microscopy were scanned and aligned by the scale‐invariant feature transform algorithm. 3D reconstruction of the skin tissue was generated using Mimics software. Fibre content, porosity, average pore diameter and specific surface area of dermis were analysed using the ImageJ analysis system. The root mean square error and mutual information based on the scale‐invariant feature transform algorithm registration were significantly greater than those based on the manual registration. Fibre distribution gradually decreased from top to bottom; while porosity showed an opposite trend with irregular average pore diameter distribution. A specific surface area of the dermis showed a ‘V’ shape trend. Our data suggested that 3D reconstruction of human skin tissue based on large‐scale serial sections could be a valuable tool for providing a highly accurate histological structure for analysis of skin tissue. Moreover, this technology could be utilized to produce tissue‐engineered skin via a 3D bioprinter in the future.     

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.     

Estimating volumes from common carp hepatocytes using design‐based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5–7 μm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three‐dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20–40 μm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single‐section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two‐dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single‐section disector. Strong correlations existed between profile‐derived versus 2D‐nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D‐nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.     

Chloroplast ultrastructure in leaves of Urtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration

In this article, we report on the adaptation of high-pressure freezing and freeze-substitution (HPF-FS) for ultrastructural analysis of leaf tissue with special emphasis on chloroplasts. To replace the gas in the intercellular spaces, a mixture of water and methanol (MeOH) was employed. We compared three different supplements for FS--osmiumtetroxide, uranyl acetate, and safranin--with regard to the preservation of the ultrastructure of chloroplasts and other cellular compartments. The results show that (i) replacement of air within intercellular spaces by 8% (v/v) MeOH has no influence on the ultrastructure of the chloroplasts, (ii) undulation of membranes frequently observed after conventional preparation of specimens does not occur during chemical fixation but during room temperature dehydration, and (iii) uranyl acetate or osmium tetroxide employed during FS are not superior over safranin.     

Design-based estimation of surface area in thick tissue sections of arbitrary orientation using virtual cycloids

Surface area is a first‐order stereological parameter with important biological applications, particularly at the intersection of biological phases. To deal with the inherent anisotropy of biological surfaces, state‐of‐the‐art design‐based methods require tissue rotation around at least one axis prior to sectioning. This paper describes the use of virtual cycloids for surface area estimation of objects and regions in thick, transparent tissue sections cut at any arbitrary (convenient) orientation. Based on the vertical section approach of Baddeley et al., the present approach specifies the vertical axis as the direction of sectioning (i.e. the direction perpendicular to the tissue section), and applies computer‐generated cycloids (virtual cycloids) with their minor axis parallel to the vertical axis. The number of surface‐cycloid intersections counted on focal planes scanned through the z‐axis is proportional to the surface area of interest in the tissue, with no further assumptions about size, shape or orientation. Optimal efficiency at each x–y location can be achieved by three virtual cycloids orientated with their major axes (which are parallel to the observation planes) mutually at an angle of 120°. The major practical advantage of the present approach is that estimates of total surface area (S) and surface density (S_V) can be obtained in tissue sections cut at any convenient orientation through the reference space.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

Effect of indirubin‐3‐monoxime against lung cancer as evaluated by histological and transmission electron microscopic studies

The aim of this study is to evaluate the antitumor effect of indirubin‐3‐monoxime and its mode of action in benzo(α)pyrene [B(α)P] induced lung cancer in A/J mice. Light microscopic examination of lung sections of [B(α)P] induced lung cancer mice revealed the presence of adenocarcinoma characterized by extensive proliferation of alveolar epithelium and loss of alveolar spaces. The control lung tissue showed a normal architecture with clear alveolar spaces. Interestingly the indirubin‐3‐monoxime treated groups showed the reduced adenocarcinoma with appearance of alveolar spaces. Transmission Electron Microscopic (TEM) studies of lung sections of [B(α)P] induced lung cancer mice showed the presence of phaemorphic cells with dense granules and increased mitochondria. The lung sections of mice treated with indirubin‐3‐monoxime showed the presence of shrunken, fragmented, and condensed nuclei implying apoptosis. The effects were dose dependent and prominent in 10 mg/kg/5 d/week groups suggesting the therapeutic role of indirubin analogue against this deadly human malignancy. Here, our results indicate that indirubin‐3‐monoxime brings antitumor effect against [B(α)P] induced lung cancer by its apoptotic action in A/J mice. Microsc. Res. Tech. 73:1053–1058, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Micromorphology,pharmacognosy, and bio‐elemental analysis of an important medicinal herb: Verbascum thapsus L.

The present study was conducted on characterization of morpho‐anatomical, phytochemical, and bio‐elemental analysis of root, stem, and leaf of Verbascum thapsus. Morphologically Verbascum is a biennial plant that flowers for a month and a half in mid‐ to late summer. Various organoleptic features of root, leaf, and stem were recorded. Anatomically the T. S of the root, stem, and leaf showed a typical dicot histological differentiation. Leaf possessed anomocytic stomata, crescent shape vascular bundles, and covered with long and stellate type trichomes while, stem contained collateral type of vascular bundles and a well‐developed pith to store phytochemicals responsible for various pharmacological activities. The powder drug study through scanning electron microscopy revealed the presence of various types of tissues. Branched, tree like and stellate trichomes in root and leaf help in absorption and reduce loss of water. These anatomical features are responsible for the survival of the plant as biennial. Four macro elements (Na, K, Ca, and Mg) and seven microelements (Cr, Cu, Fe, Mn, Ni, Zn, and Cd) and their concentrations in ppm were also studied using Atomic Absorption Spectroscopy. Phytochemical screening of methanolic extract showed existence of various secondary metabolites, while mucilage and anthraquinones was not detected. The present study helps to understand the taxonomic identification of the plant based on morpho‐anatomical features and throws the attention of the researchers to carry out the work for developing its various formulations, which can ultimately be beneficial for the human beings as well as animals.     

Vertical LM sectioning and parallel CT scanning designs for stereology: application to human lung

Practical, unbiased stereological methods are described to estimate lung volume and external surface area, and total volume and surface area of relatively large and anisotropic structures (bronchi and arteries) inside the lung. The volume of each of five lung strata was estimated first by fluid displacement and then by computed tomography (CT) using Cavalieri's method; the reliability of CT was assessed through a calibration procedure, and image thresholding criteria for an accurate volume estimation using CT were established. The parallel, perfectly registered CT section images were also used to estimate the external surface area of each stratum by the spatial grid method. Unbiased estimation of internal surface areas in lung is a long-standing problem: since the structures are large and essentially void, large sections are needed; to facilitate identification, thin sections have to be used for light microscopy, and since such structures are anisotropic, the sections should be vertical. A practical stereological design is demonstrated here on an infant lung, which fulfils all these requirements. This study illustrates the potential of using unbiased stereology to characterize infant pulmonary hypoplasia.     

3D analysis of synaptic vesicle density and distribution after acute foot‐shock stress by using serial section transmission electron microscopy

Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter‐containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot‐shock stress and (2) rats with sham stress as control group. Two‐dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in‐section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three‐dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one‐dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot‐shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.     

Vitreous cryo-sectioning of cells facilitated by a micromanipulator

Sectioning vitrified cells and tissues for cryo‐electron microscopy is more challenging than room‐temperature sectioning of plastic‐embedded samples. As the sample must be kept very cold (相似文献