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.     

Maternal dexamethasone treatment reduces ovarian follicle number in neonatal rat offspring

Elevated glucocorticoid levels in the gravid female circulation affect a number of endocrine functions in the fetuses and neonates. The aim of this study was to examine the effects of maternal dexamethasone (Dx) administration during late pregnancy on the ovaries of neonatal offspring. On the 16th day of pregnancy, experimental dams received subcutaneously 1.0 mg Dx/kg b.w., followed by 0.5 mg Dx/kg b.w./day on the 17th and 18th days of gestation. The control gravid females received the same volume of saline vehicle. Left ovaries from 5‐day‐old female pups were stereologically analyzed. The ovary volumes were estimated using Cavalieri's principle. The number of healthy and atretic primordial and primary follicles was estimated using a fractionator–physical disector method. The number of secondary follicles was determined by exact counts of every fourth section encompassing whole cross‐sections of the ovary. The ovary volume was significantly decreased (by 44.4%; P < 0.05) in the group of female pups from Dx‐treated mothers comparing to the controls. The numbers of healthy primordial and atretic follicles were 38.8% (P < 0.05) and 50.9% (P < 0.05), respectively, reduced in the ovaries of pups from the Dx‐treated mothers, when compared with the control values. There were 53.4% (P < 0.05) fewer healthy primary and 41.8% (P < 0.05) fewer healthy secondary follicles as well. The numbers of atretic primary and secondary follicles were reduced by 60.0% (P < 0.05) and 61.7% (P < 0.05), respectively. It can be concluded that fetal exposure to glucocorticoids decreased the pool of non‐growing follicles in the neonatal ovary, whereas the processes of folliculogenesis and atresia remained unaffected.     

High-Resolution scanning electron micrographs of freeze-cracked cells in testes from normal and irradiated rats at different stages of gonadal development

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.     

Histology and ultrastructure of the testis and vas deferens in Pseudochorthippus parallelus parallelus (Orthoptera,Acrididae)

Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) is a widespread species in Europe, and also it is localized in some regions in Turkey such as Bursa, Eski?ehir, Ankara, Bolu, Düzce, and Çank?r?. The features of the reproductive organs such as the numbers and shapes of testes and follicles can be used as taxonomical characters. For this purpose, the ultrastructural and histological features of testis and vas deferens in P. parallelus parallelus were examined with using light microscope, scanning electron microscope, and transmission electron microscope. The mature P. parallelus parallelus has two conjugated testes produce spermatozoa. Each testis is composed of numerous testis follicles in which different stages of spermatogenesis and spermiogenesis develop. First, spermatocytes are formed by the mitosis division of the germ cells at the distal end of the follicles. Then, spermatocytes form spermatids by meiosis division in the middle region of the follicles. Finally, spermatids are differentiated to spermatozoa at the proximal region of the follicles. After maturation of the spermatozoa, sperm tails come together as the sperm bundles called as spermatodesm. Each follicle is connected to vas deferens via vas efferens to discharging spermatozoa. In spite of some differences, the testes and the vas deferens in P. parallelus parallelus are highly similar to the those of other species, especially Orthopteran species.     

A simple technique to measure the movements of the microscope stage along the x and y axes for stereological methods

Measurements of microscope stage movements in the x and y directions are of importance for some stereological methods such as the optical disector and optical fractionator. The length of stage movements can be measured with great precision and accuracy using a suitable motorized stage, which is generally a computer-assisted instrument. This type of equipment is generally too expensive for and not readily available in many laboratories. This paper describes a simple method to measure the movements of the microscope stage along the x and y directions, which can be used for purposes such as systematic uniform random sampling. It needs a microscope attachment consisting of two dial indicators; one of them is used to measure the amount of stage movement along the x -axis and the other measures the amount of movement along the y -axis. Movements of the stage on the micrometre-scale can be measured easily using this device.     

Application of the disector method in the light microscopic quantification of type II pneumocytes in control and ozone-exposed rats

Alveolar type II cells in control and ozone-exposed rat lungs were counted at the light microscopical level with the ‘disector method’. The type II cells were unequivocally marked by histochemical staining for alkaline phosphatase activity in 2 μm plastic sections. By this counting method, the mean number of type II cells per lung in control rats was of the same magnitude as those reported in the literature, using point counting methods. After exposure of rats to 1.6 mg ozone/m³ for 7 days, a 50% increase in the mean number of type II cells was observed. The use of the disector method at the light microscopical level offers some advantages above a quantification at the electron microscopical level. The procedure is less time-consuming, larger areas can be screened, two parallel countings can be performed in one set of sections and there is no need for an exact knowledge about the diameter of the measured particle.     

Estimation of the section thickness and optical disector height with a simple calibration method

The distance between the upper and lower surfaces of a section (i.e. the section thickness) can be measured with a microcator or a shaft encoder. In the present report, an alternative simple method is described for estimating the section thickness where such equipment is not available. The basic principle of the method is based on a calibration method already described in the literature. The main difference is that it enables one to make more precise measurements. Provided that the calibration and measurements are made properly, this method can be used in estimating the section thickness, optical disector heights, and in particular in the determination of the thickness sampling fraction for the optical fractionator.     

Is there section deformation resulting in differential change of nuclear numerical densities along the z axis of thick methacrylate or paraffin sections?

The optical disector, a three‐dimensional counting frame or probe in stereology, is often positioned in the middle (depth) of a thick section for unbiased nuclear counting. Using 30–40 μm thick methacrylate or paraffin sections for nuclear counting of neurons with the optical disector, however, some studies showed markedly higher nuclear densities at 10% of the section thickness near the top or bottom surface of the section, suggestive of deformation of section along its z axis and thus affecting the number estimation. To verify the findings, this study obtained two sets of 12–14 methacrylate sections (average thicknesses 21.7 and 29.4 μm) and two sets of 12 paraffin sections (average thicknesses 13.8 and 29.2 μm) from mature rat testes. Each section was used to count round spermatid nuclei in the seminiferous epithelium densely packed with the cells, using 3–4 consecutive disectors placed vertically (along the z axis of the section) from the top surface of the section, through the whole section thickness (two sets of methacrylate and paraffin sections) or in 80–83% of the thickness (other sections). The results demonstrated that, overall, there were no considerable nonuniform changes of the nuclear densities along the z axis of the sections.     

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.     

Characterization of nickel silicides using EELS-based methods

We present an unbiased estimator of the total number of alveolar structures distal to the transition from a bronchiole to an alveolar duct system (‘ventilatory units’, VUs). In species without respiratory bronchioles, including mice, the number of VUs is equivalent to the number of acini. The acinus is a functional unit of gas exchange, defined as a parenchymal unit distal to a terminal bronchiole in which all airways contain alveoli and thus participate in gas exchange. The estimator combines two different estimators of the number of VUs: (1) an estimator derived from the Euler number of all the openings of the bronchial tree and (2) an estimator derived from direct counts of topological changes occurring at bronchiole‐alveolar duct junctions. Combining the two estimators eliminates the requirement to be able to identify even vanishingly small pieces of bronchial tissue in physical disectors. We implemented the fractionator estimator in five adult mice lungs using physical fractionators with varying but known sampling fractions (Horvitz–Thompson estimator). We obtained total values of about 4200 VUs (CV = 0.05) in 21‐day‐old and 4480 (CV = 0.06) in 69‐day‐old animals. Being fractionator estimates, these total numbers are independent of shrinkage. The densities of VUs per unit volume of tissue (values corrected for tissue shrinkage) were similar in left and right lungs.     

Pronounced loss of cell nuclei and anisotropic deformation of thick sections

The local deformation and variations in section thickness are studied in 100-μm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 μm and the lower 10 μm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.     

Stereological length estimation using spherical probes

Lineal structures in biological tissue support a wide variety of physiological functions, including membrane stabilization, vascular perfusion, and cell‐to‐cell communication. In 1953, Smith and Guttman demonstrated a stereological method to estimate the total length density (L_v) of linear objects based on random intersections with a two‐dimensional sampling probe. Several methods have been developed to ensure the required isotropy of object–probe intersections, including isotropic‐uniform‐random (IUR) sections, vertical‐uniform‐random (VUR) slices, and isotropic virtual planes. The disadvantages of these methods are the requirements for inconvenient section orientations (IUR, VUR) or complex counting rules at multiple focal planes (isotropic virtual planes). To overcome these limitations we report a convenient and straightforward approach to estimate L_v and total length, L, for linear objects on tissue sections cut at any arbitrary orientation. The approach presented here uses spherical probes that are inherently isotropic, combined with unbiased fractionator sampling, to demonstrate total L estimation for thin nerve fibres in dorsal hippocampus of the mouse brain.     

Stereology for anisotropic cells: Application to growth cartilage*

A number of either new or recently available stereological methods are described for estimating volume, surface area and number of anisotropic cells. The methods are illustrated with direct reference to the epiphyseal growth plate. Different estimates of a given quantity are obtained by applying alternative methods to the same set of sections, in order to compare the relative merits of the methods. For instance, the surface area of the cells is estimated via the Dimroth–Watson model (which gives a measure of the degree of anisotropy in addition to the surface area estimate) and from vertical sections using cycloid test systems. Cell number is estimated by traditional unfolding methods and by the new disector method. Also, volume-weighted mean cell volume is estimated from vertical sections via point-sampled intercepts using two different kinds of rulers to classify intercept lengths. Finally, nested design statistics is applied to a set of data from twelve animals in order to compare the relative impacts of biological and stereological (sampling) variations on the observed coefficient of error of a group mean estimate. The preferred methods are listed in the final section.     

Organization and seasonal quantification of the intertubular compartment in the bat Molossus molossus (Pallas, 1776) testis

Environmental factors can influence the reproductive rates in bats, and since morphometric information of bats testis is scarce, we aimed to compare the organization and quantification of the intertubular components in the testes of the bat Molossus molossus, collected in different seasons. Testicular histological sections were evaluated using light and electron microscopy. The intertubular compartment occupied an average 10% of the testes, being predominately constituted of Leydig cells (LC). The percentages of the testes occupied by the intertubular compartment and by LC were significantly higher in summer, while the other intertubular components did not vary significantly among the seasons. As suspected under light microscopy, the ultrastructural analysis confirmed the existence of multinucleated LC during winter. The increase in the nuclear percentage of LC in winter seems to have caused the decrease of the cytoplasmatic measurements in that season, as well as in the volume of LC. The highest cytoplasmatic values and volume of LC registered in the spring, summer, and fall can be related to greater activity of this cell in these seasons. The higher investment in intertubular tissue and in LC observed in summer, compared to winter; suggest an increase in the steroidogenic capacity of this bat during summer. The analyses correlating testicular morphometry and abiotic environmental factors in this study confirm the influence of climatic factors on the reproduction of M. molossus. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Near-infrared optical spectroscopy for pancreas shrinkage estimation with multi synchrosqueezing transform and multivariate regression model

In this article, we proposed a method to estimate pancreas shrinkage with pancreas β cell insulin secretion. The β cells in the pancreas secrete insulin and digestive enzymes after food consumption. Conventionally, the pancreas structure estimation is done with magnetic resonance imaging (MRI) and ultrasound imaging techniques. However, the structure of the pancreas changes due to islet cell death. The presence of islet cells is detected through near infrared (NIR) spectroscopy signal acquired from the epigastric region (pancreas) of the abdomen. Subsequently, the NIR spectroscopy signal from the pancreas is analyzed with multi synchrosqueezing transform (MSST); whereas, the β cell insulin secretion varies for diabetic and nondiabetic persons. The existence of β cell and insulin secretion correlates with Root Mean Square (RMS) and kurtosis via a multivariate regression model to evaluate pancreas shrinkage. In terms of numerical results, NIR spectroscopy signal from the pancreas was obtained for about 20 nondiabetic and 20 diabetic persons. The pancreas shrinkage was estimated with 88% accuracy. The results are validated with MRI pancreas images for earlier detection of the apoptotic pancreas. The pancreas shrinkage causes lower insulin emission and unpredictable blood glucose in diabetic patients. Analysis of NIR spectroscopy signals of the pancreas with MSST was done to obtain higher-order and lower-order frequency components.     

Unbiased stereological estimation of the rat fetal pituitary volume and of the total number and volume of TSH cells after maternal dexamethasone application

Glucocorticoids have an inhibitory influence on proliferation activity of the pituitary cells while stimulating apoptosis. Therefore, it was hypothesized that the synthetic glucocorticoid, dexamethasone (DX), has an inhibitory influence on the number of thyroid‐stimulating hormone (TSH) cells during fetal development. The effects of maternal administration of DX on stereological parameters of TSH cells, and TSH serum concentration were investigated in 21‐day‐old rat fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg DX/kg b.w. subcutaneously, followed by 0.5 mg DX/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline vehicle. TSH cells were stained immunocytochemically by the peroxidase–antiperoxidase (PAP) method. The fetal pituitary volumes were estimated using Cavalieri's principle. A physical disector counting technique in combination with the fractionator sampling method was used for estimation of pituitary TSH cell number. Cell and nuclear volumes were measured with a planar rotator. Maternal DX application was found to cause a significant decrease of pituitary volume and number of TSH cells per pituitary in 21‐day‐old fetuses in comparison with the control fetuses. TSH cell number expressed per body weight unit declined significantly after maternal DX administration. These results indicate an inhibitory DX influence on proliferative activity of precursors and likely differentiated TSH cells and increased apoptotic prevalence. The histological appearance, volume of TSH cells and TSH serum concentration suggest intensive synthetic activity in TSH cells of DX exposed fetuses. Microsc. Res. Tech. 73:1077–1085, 2010. © 2010 Wiley‐Liss, Inc.     

Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope

A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 μm thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.     

Analysis of shrinkage and warpage in an injection-molded part with a thin shell feature using the response surface methodology

This paper presents a systematic methodology to analyze the shrinkage and warpage in an injection-molded part with a thin shell feature during the injection molding process. The systematic experimental design based on the response surface methodology (RSM) is applied to identify the effects of machining parameters on the performance of shrinkage and warpage. The experiment plan adopts the centered central composite design (CCD). The quadratic model of RSM associated sequential approximation optimization (SAO) method is used to find the optimum value of machining parameters. One real case study in the injection molding process of polycarbonate/acrylonitrile butadiene styrene (PC/ABS) cell phone shell has been performed to verify the proposed optimum procedure. The mold temperature (M _T), packing time (P _t), packing pressure (P _P) and cooling time (C _t) in the packing stage are considered as machining parameters. The results of analysis of variance (ANOVA) and conducting confirmation experiments demonstrate that the quadratic models of the shrinkage and warpage are fairly well fitted with the experimental values. The individual influences of all machining parameters on the shrinkage and warpage have been analyzed and predicted by the obtained mathematical models. For the manufacture of PC/ABS cell phone shell, the values of shrinkage and warpage present the reduction of 37.8 and 53.9%, respectively, using this optimal procedure.     

Determination of the mean caliper diameter of lung nuclei by a method which is independent of shape assumptions

When determining the number of nuclei (therefore cells) present in a known volume of tissue it is necessary to determine the overall mean caliper diameter of nuclei for each cell type studied. Most previous methods for determining the mean caliper diameters (D?) of cell nuclei were strongly dependent upon assumptions of the shape of the particles in question (Greeley et al., 1978). A computer program has now been designed to calculate D? from three-dimensional reconstructions of nuclei in a manner that is independent of all shape assumptions. The D?s of ten capillary endothelial cell nuclei from normal rat lungs have been determined by this shape-independent method. The resulting overall mean caliper diameter for this group of ten nuclei varied only slightly from that previously estimated for them by a method which assumes them to be triaxial ellipsoids. In a similar comparison, the D?s for ten normal human lung endothelial cell nuclei were determined by both methods. The overall mean caliper diameter determined by the shape-independent method was 10% larger than that estimated by the shape-dependent method. This suggests that human lung endothelial nuclei vary significantly from the shape assumption (triaxial ellipsoid) employed in the shape-dependent method, and the shape-independent method was considered to yield a more accurate measurement of D?.