Intersection Counting Research Articles

Surface exchange area of the porcine placenta: morphometry of anisotropic interdigitating microvilli.

The pig has a diffuse epitheliochorial placenta. The foeto-maternal surface area (Sv(F), Sv(M)) is enlarged at three levels: level I: macroscopic folds of uterine mucosa covered by foetal membranes; level II: light microscopic folds of uterine epithelium covered by trophoblastic epithelium; level III: ultrastructural formation of both maternal and foetal microvilli in an interdigitating pattern. This study presents a morphometric method, using intersection counting, to estimate the surface area of these interdigitating, highly oriented microvilli at level III, i.e. the 'true' surface exchange area. The method is applied to different stages in pregnancy. The epithelial surfaces are enlarged by a factor 8-11 due to the formation of microvilli. This factor varies during pregnancy, being lower early in gestation and before delivery, and for the first time it is shown to be significantly different for mother and foetus. At level III the foetal surface of placenta is larger than the maternal indicating that the two opposing surfaces are not congruent at the ultrastructural level and that membrane-linked placental transport may be different in mother and foetus. A three-compartment model is suggested for placental exchange with maternal, intermicrovillous and foetal compartments.

Timing of the quantitative recovery in the regenerating rat lung.

Growing rats 23 days of age were subjected to the resection of the upper and middle lobes of the right lung (25% of total lung volume). On postoperative days 1, 4, 6, 9, 12, 18, and 30, the lungs of 5 animals were fixed by intratracheal instillation of glutaraldehyde, their volumes measured by water displacement, and the lung tissue processed for quantitative light (LM) and electron microscopic (EM) investigations. For each group, 3 age-matched sham-operated, and 4 normal rats served as controls. Sham operation consisted in entering the pleural space, and collapsing and ventilating the lungs. In all animals, the following parameters were measured by means of point and intersection counting stereology at either LM or EM-levels: volume densities of lung parenchyma and of its components (airspaces, tissue, capillary blood) and surface densities of airspaces and of capillaries. From these data absolute parameter values were calculated for each lung. The results showed that after bilobectomy the remaining lung re-expanded rapidly first by an overinflation of the airspaces and after Day 4 by an increase in tissue mass and capillary volume. On Days 9 and 12 the operated lungs did not differ quantitatively from control lungs. Later, however, further sequels of the bilobectomy were detected: On Day 18, lobectomy-lungs were smaller than controls, and on Day 30 lungs, the left lung was significantly different in structure from the right lung and from control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

The problem of ambiguous profiles of microvilli between apposed cell surfaces: a stereological solution.

When interdigitating microvilli projecting from apposed cell surfaces are sectioned randomly, it becomes impossible to specify to which of the two parent surfaces an individual microvillous profile belongs. Because of this image ambiguity, direct estimation of the ratio of microvillous surface areas by intersection counting cannot be performed. A stereological solution to this problem is proposed. It relies on counts of the numbers of bases of microvilli identified on each reference (parent) surface.

Neurotransmitters of the hypothalamic suprachiasmatic nucleus: Immunocytochemical analysis of 25 neuronal antigens

An immunocytochemical analysis with 33 antisera was undertaken to investigate the localization of 25 different neurotransmitter-related antigens in the hypothalamic suprachiasmatic nucleus in the rat. To obtain estimates of relative densities of immunoreactive axons a stereological approach was used involving counting of intersections of immunoreactive axons with a superimposed semi-circle test grid. All neurotransmitter-related antigens found in perikarya within the suprachiasmatic nucleus, including those stained with antisera against bombesin, gastrin-releasing peptide, neurophysin, vasopressin, somatostatin, γ-aminobutyrate, glutamate decarboxylase and vasoactive intestinal polypeptide were also found in axons within the nucleus. A greater number of these immunoreactive axons was found within the nucleus than in the adjacent anterior hypothalamus. The size of all immunoreactive axons in the suprachiasmatic nucleus was consistently small; immunoreactive axons were found ramifying widely in the nucleus, often ending with terminal boutons near perikarya immunoreactive for the same antigen. All neurotransmitter-related substances found in perikarya of the suprachiasmatic nucleus were also found in axons crossing over the midline to innervate the contralateral nucleus, providing an anatomical substrate for a high degree of communication between the paired nuclei. Axons immunoreactive for other putative transmitters including serotonin arising outside the nucleus were also found in high densities within the nucleus and crossing over the midline between the nuclei. Immunoreactivity for some transmitters was found in axons of similar densities within and outside the nucleus, including antisera against tyrosine hydroxylase; a small number of dopamine β-hydroxylase and a few phenylethanolamine N-methyltransferase-immunoreactive axons were found in the SCN, suggesting that dopamine, norepinephrine and epinephrine may occur in a limited number of axons in the nucleus. Small numbers of axons immunoreactive with antisera raised against cholecystokinin, prolactin, substance P, thyrotropin-releasing hormone and choline acetyltransferase were found within the suprachiasmatic nucleus. Axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, α-melanocyte-stimulating hormone and neurotensin were rarely found within the suprachiasmatic nucleus; axons immunoreactive for luteinizing hormone-releasing hormone, adreno-corticotropic hormone, cholecystokinin and tyrosine hydroxylase were found in both horizontal and coronal sections in the area between the left and right suprachiasmatic nuclei. Tyrosine hydroxylase-immunoreactive cell bodies were situated in the same midline area, as well as at the lateral and dorsal borders of the suprachiasmatic nuclei. Although axons immunoreactive for any one substance were not found exclusively in any one area of the nucleus, regional differences in axon density were found; bombesin- and gastrin-releasing peptide-immunoreactive axons, for instance, were more frequent in the ventral suprachiasmatic nucleus near immunoreactive perikarya, while vasopressin-neurophysin immunoreactivity appeared greater in the dorsomedial part of the nucleus. Efferent projections from the nucleus to hypothalamic and extrahypothalamic areas are discussed and the probable cross-reaction of different antisera with a single antigen is considered. The finding of many different putative neurotransmitters within the suprachiasmatic nucleus serves to underscore the complex axonal organization of the nucleus.

A quantitative ultrastructural study of alterations in the area of the basal cell-stromal interface during experimental oral carcinogenesis.

Because of its topographical location, the basal cell in carcinomas must play a significant role in the invasion of adjacent tissues. We have analysed the proportion of the basal cell plasma membrane in direct contact with the adjacent lamina propria during in vivo oral carcinogenesis. Samples of hamster cheek-pouch mucosa treated with DMBA were assigned to hyperplasia, dysplasia and carcinoma groups using strictly defined histopathological criteria. Electron micrographs of basal cells were subjected to stereological intersection counting to provide estimates of the proportion of the total basal cell membrane in contact with the connective tissue (SSBM,PM). In untreated controls, a mean value of 9% was obtained, whereas, for DMBA-induced hyperplasias, dysplasias and carcinomas, values were 8%, 12% and 16% respectively. Statistically, SSBM,PM values were significantly elevated in dysplasias and carcinomas. The changes in this parameter may reflect an increased motility in the transforming basal cell prior to and concomitant with cellular invasion, and may prove to be of value as a structural indicator of malignant transformation.

On the Stereological Estimation of Numerical Density of Particle Systems by an Object Counting Method

AbstractThe determination of numerical density Nv of particles is one of the most complex problems in stereology. This paper presents a method for solving it, consisting only of counting of intersections between particles and thick slices of some different thicknesses. The essential stereological parameters Nv and mean caliper diameter D of the particles are determined by means of a regression line. The basic formula is a simple consequence of general principles for deriving stereological formulae which is described in this paper. The object counting method is applicable if the particles are convex and if the whole particle system can be described by a stationary isotropic ergodic marked point process. A practical example (liver cell nuclei of rats) demonstrates the application of the method.

Morphometry in Pathology: Another Look at Diagnostic Histopathology

Histopathology is crucial for diagnostic and therapeutic decisions in many disease states. The created classifications, however, have not always allowed explicit recognition of diagnostic categories. So, considerable intra- and interobserver variations are possible under diagnostic circumstances. Better reproducibility can be reached by applying other than traditional methods. These include morphometry as well as special stains, electron microscopy and immunohistochemistry. Morphometry is basic in that in detects structural and morphological aberrations in samples prepared by other methods and without doubt brings the element of accuracy to support the diagnostic decision. Morphometric approach includes item classification and grading, point counting and intersection counting methods, and the use of various semiautomatic or automatic instruments. In statistical morphometry morphometrical parameters are collected from several disease cases. Such data reinforced with prognostic follow-ups are the basis for disease classification. Diagnostic morphometry, on the other hand, tries to study the sample of one individual and give relevant data for diagnostic decisions. Morphometric methods allow estimation of the limits of possible performance in histopathology, estimation of human performance in the diagnostic process and estimation of the influence of interfering human factors. Diagnostic decisions as well as decisions linked with therapy are basically probabilistic. Also the data collected by diagnostic morphometry should be linked with probabilistic estimates of the prognostic relevance of the findings. Education of histopathology should be developed to meet these demands, both at elementary medical level and at postgraduate medical level. Education of morphometry can be incorporated into the medical curricula as shown by the experience at the University of Kuopio where aspects of morphometry are taught during histopathology course and special postgraduate courses have been arranged.

Geometric model of the rat intestinal mucosa for stereological evaluation of villus amplification factors.

This study was prompted by a desire to compare villus amplification factors at various sites along the small intestine of the laboratory rat. The paper describes a precise and efficient stereological method for achieving this objective. The method relies on conventional intersection counts performed exclusively on transverse sections. Analyses are based on six adult rats. Pieces of small bowel fixed by in situ perfusion were taken from proximal, middle and distal regions. Intersection counts on villus and primary mucosal surfaces were performed on transverse and longitudinal sections through the same regions. These counts revealed that intestinal architecture can be reasonably approximated by the simple geometric model of an isotropic villus surface on an anisotropic (cylindrical) primary mucosal surface. As a result, intersection counts may be confined to transverse sections (to reduce sampling variance) and the appropriate ratio estimator multiplied by the numerical coefficient 4/pi (to obtain an estimate of surface amplification due to villi).

Ultrastructural morphometric of gap junctions during differentiation of stratified squamous epithelium.

The presence of gap junctions in stratified epithelia has now been extensively documented, but there have been few attempts to quantify them. In the present report, samples of hamster cheek pouch mucosa were processed for electron microscopy and electron micrographs from defined basal, spinous and granular layers were obtained. Using a combination of direct measurement and stereological intersection counting techniques, the relative surface areas of peripheral gap junctions (i.e. those in direct contact with the epithelial plasma membrane) and annular gap junctions (i.e. those present as complete, approximately circular profiles within the epithelial cell cytoplasm) were determined. Following estimation of the plasma membrane surface area of 'average' epithelial cells from each of the defined strata, relative values were transformed into absolute data. Data from peripheral and annular junctions were pooled to provide an estimate of total gap junctions area. Relative surface area estimates were similar for peripheral, annular and total gap junctions, in that values were invariably highest in the spinous layer and lowest in the granular layer. Absolute data indicate that there is more than a threefold increase in the area of membrane differentiated into gap junctions in the average spinous cell when compared with the average basal cell. Values for total gap-junctional areas in the average granular cell are reduced somewhat with respect to the average spinous cell and this is effected by a decrease in the area of peripheral gap junctions. We conclude that there is synthesis of gap junctions between basal and spinous cells, which is followed by evidence of degradation between spinous and granular cells. The magnitude of the estimates of area is comparable to those obtained from other stratified and non-stratified epithelia and it would thus appear that gap junctions may play a significant role in cellular control processes in all viable epithelial strata.

Desmosomes in hamster cheek pouch epithelium: their quantitative characterization during epithelial differentiation.

Desmosomes in stratified squamous epithelia appear to exhibit quantitative alterations during differentiation. In this work we use stereological and other morphometric methods to quantify these structures in epithelial cells from defined basal, spinous and granular strata. Hamster cheek pouch mucosa from five animals was processed for electron microscopy using strictly standardized techniques and a stratified random sampling procedure was used to obtain micrographs of cells from basal, spinous and granular layers. Stereological intersection counting techniques were used to determine for each layer the relative surface area of plasma membrane occupied by desmosomes (Ss), the number of desmosomes per unit surface area of plasma membrane (Ns), the mean individual desmosomal diameter (delta) and the mean individual desmosomal surface area (s). In addition, estimates of nuclear volume were obtained by direct measurement of nuclear profiles and volume-to-surface ratios were obtained by a combination of point and intersection counting, which enabled estimates for the volume (Vcell) and plasma membrane surface area (SPM) of the 'average' cell within each stratum to be acquired. Using this information, it was then possible to calculate both the total surface area (S) and the number (N) of desmosomes on the plasma membranes of average cells. The parameters Ss and Ns showed progressive increases between basal and granular layers, whereas values for delta and s were lower in granular cells when compared with basal and spinous cells. The parameters Vcell, SPM, S and N all increased progressively and significantly during differentiation. Between basal and granular layers, the mean cell volume and surface area had each increased approximately threefold, whereas the surface area and number of desmosomes on the average cell plasma membrane had increased approximately seven- and eleven-fold, respectively. Granular cells thus possess more numerous desmosomes, which occupy a greater proportion of the plasma membrane area but which are individually smaller, when compared with basal and spinous layers.

Morphometrics of the avian lung. 3. The structural design of the passerine lung

The lungs of 46 adult, wild passerine birds belonging to 8 species have been analysed morphometrically, both by light and electron microscope. Volumes were estimated by point counting, surface areas by intersection counting, and thicknesses by intercept length measurements. The mean values obtained for these passerine species appertaining to both lungs together were: volume of the lung per kilogram body weight 25 cm 3/kg, volume density of the exchange tissue 52% surface area of the 0 blood-gas (tissue) barrier per gram body weight 47.48 cm 2/g, surface density of the blood-gas (tissue) barrier 323.8 mm 2/mm 3, capillary loading 1.15 cm 3/m 2, harmonic mean thickness of the blood-gas 0772 (tissue) barrier 0.127 μm, arithmetic mean thickness 0.745 μm and the total morphometric pulmonary diffusion capacity 7.08 ml O 2/min/mm Hg/kg. These values indicate that the passerine lung is specially well adapted for gas exchange, mainly by having a thin and extensive blood-gas (tissue) barrier, in response to the high oxygen demand by this group of bird.

Hemidesmosomal dimensions and frequency in experimental oral carcinogenesis: a stereological investigation.

Connective tissue invasion by carcinomas results in a variety of morphological changes at the epithelial-connective tissue junction. In this report, hemidesmosomal size (-delta) and frequency (Ns) were quantified using established stereological methods. Following treatment of hamster cheek pouches with the chemical carcinogen DMBA, lesions defined as hyperplasias, dysplasias and carcinomas were evaluated using electron microscopical techniques. Untreated pouches served as controls. Stereological intersection counting procedures were used to quantify hemidesmosomes at the epithelial-connective tissue junction. During carcinogenesis, hemidesmosomal diameter decreased progressively, although the reductions were not statistically significant. Hemidesmosomal frequency also decreased progressively between control and carcinoma groups. Normal basal plasma membranes possessed approximately 8 hemidesmosomes/micron 2 whereas values were reduced to approximately 3/micron 2 in the carcinoma group. The reductions in hemidesmosomal frequency during carcinogenesis may reflect an increased motility of basal epithelial cells and a decreased adherence of basal cells to the adjacent lamina propria. The specificity of this alteration in the pathogenesis of malignancy can be readily determined by the application of stereological methods to various neoplastic and non-neoplastic lesions.

Stereological determination of structural anisotropy

A general mathematical formulation is given to the problem of determining the structural anisotropy by means of the stereological principle. Three cases are considered—distributed curves in the plane, distributed curves in the space and distributed surfaces in the space. The number of intersections with a probe line or plane is viewed as a transformation, which is termed the “Buffon transform”, between two distribution densities, and a form of its inverse transform is given. Then, the change of anisotropy due to the deformation of the material is formulated, and the strain is shown to be determined from the data of the intersection counting. All equations are written in the form of Cartesian tensor equations invariant to coordinate translations and rotations. A typical example is also given.

Automated techniques for the study of lung alveolar stereological parameters with the IBAS image analyser on optical microscopy sections.

In the case of lung alveoli, the good contrast of the features allows the use of a fully automated image analysing procedure, yielding many of the desirable stereological parameters which can be estimated on optical microscopy slides: alveolar volume and surface densities, a form factor and average mean integral curvature. With our program, specially designed for the IBAS image analyser (Kontron Bildanalyse, Munich, B.R.D.), the whole automated process takes an average of 28 s per field. The image analyser-obtained stereological estimates compare well with those given by point and intersection counting methods. Some difficulties inherent in image analysers arise when measuring alveolar boundary lengths: a scheme is therefore proposed which eliminates the image-edge errors, with no sampling bias, by comparing three different frames inside each field. A solution to the problem of estimating automatically the positive and negative tangent counts, necessary for the curvature calculations, is also proposed. Our program has been applied routinely to the study of premature newborn rabbit lung alveoli, as part of a work dealing with an evaluation of the possibility of preventing the respiratory distress syndrome by the administration of heterologous surfactant.

Cellular Changes in Rat Urinary Bladder Carcinomas Induced by Fanft: A Quantitative Electron Microscopic Analysis

Morphometric data of normal and neoplastic urinary bladder epithelia have been collected from the Fischer rat FANFT model. Sequential measurements of volumes, surface areas, and numerical densities of organelles, and where pertinent, cellular compartments have been made during FANFT carcinogenesis, utilizing standard point and intersection counting methods. The data show that neoplastic transformation of rat bladder epithelium, and progression of FANFT tumors are associated with increasing volume densities of cells, nuclei, microvilli, rough endoplasmic reticulum, and mitochondria, as well as with decreasing volume densities of the cytoplasmic matrix, fusiform vesicles, Golgi complex, and lysosomes. Surface densities of the plasma membrane (microvillar), nuclei, rough endoplasmic reticulum, as well as mitochondrial outer and inner membranes progressively increase while surface densities of non-microvillar plasma membrane, fusiform vesicles, and Golgi complex decrease with time. Smooth endoplasmic reticulum reaches its maximum volume and surface densities in tumors present 26 weeks after the initiation of FANFT feedings (26-F tumors). This may reflect the function of smooth endoplasmic reticulum as the site of detoxification of the carcinogen, noting that FANFT is metabolized by microsomal enzymes, and in this experiment is fed only for 26 weeks. The nuclear/cytoplasmic ratio is 1:4 in normal Fischer rat urothelium, 1:3 in 26-F and 43-F tumors, which are noninvasive, and 1:2 in 61-F, which are invasive tumors. The quantitative data correlate well with the changes in degree of differentiation of the tumors and with their biological behavior.

Quantitative light and electron microscopical studies of the epithelial-connective tissue junction in intraoral mucosae.

The present study was designed to clarify the morphological nature of epithelial-connective tissue attachment by quantitative comparison of palatal and buccal mucosae. Tissue samples were obtained from ferrets and, following a strict sampling regime, sections were obtained for quantitative light and electron microscopy. Electron micrographs from the epithelial-connective tissue junction were subjected to analysis by serological intersection counting. Quantitative results show that palatal epithelium has a comparatively convoluted interface, and is twice as thick as buccal epithelium. Stereological data are presented for relative surface area of basal plasma membrane occupied by hemidesmosomes, mean hemidesmosomal diameter and numerical density of hemidesmosomes per unit area of basal plasma membrane. Larger hemidesmosomes are found in the palatal epithelium. Results suggest that a prominent rete-ridge pattern, as seen in the palatal epithelium, along with a greater specialized surface for adhesion, may be responsible for providing epithelial-connective tissue stability in regions subjected to high mechanical stress.

The use of quadrats and test systems in stereology, including magnification corrections

SUMMARYAt present a model‐free, design‐based theory of unbiased estimation, and a model‐based one of linear unbiased estimation of minimum variance, are available for stereology. The main developments rest upon the nested scheme {section (quadrat)}, whence the raw data are expressible in terms of area, length and number. The main aim of this paper is to complete the available model‐based theory by introducing the step in which sections are analysed by point‐counting via coherent test systems (CTSs). Using this development, the stereologist should be able to handle raw point and intersection counts optimally, in order to find the best estimator of a ratio R in a given specimen in a wide range of circumstances. The latter include, for instance, the use of different CTSs on different sections and of double CTSs on each section, as well as the case—(not uncommon in electron microscopy)—in which different sections from the same sample are observed at slightly different magnifications but analysed by quadrats (via automatic or semi‐automatic image analysers, for instance), or CTSs of fixed sizes. The main conclusion pertaining to the latter case is that the estimators obtained via section‐wise magnification corrections are in general superior to those corrected via a global, average magnification.In order to illustrate the methodology, a synthetic numerical example, and a real one, are given.

A quantitative study of lamina densa alterations in hamster cheek pouch carcinogenesis.

Lesions produced by topical application of 0.5 per cent. DMBA to the hamster cheek pouch epithelium were classified as hyperplasia, dysplasia and carcinoma groups using strict histological criteria. Untreated epithelium served as a control. Tissue samples from five animals in each group were processed for electron microscopy and electron micrographs from the epithelial-connective tissue junction were obtained from 5 blocks per animal. The micrographs were subjected to stereological intersection counting to determine the relative surface (SSLD, BM) of lamina densa which was in normal relationship to the basal cell plasma membranes. Quantitative results indicated a progressive loss of lamina densa during carcinogenesis and this was accompanied by the extrusion of pseudopodia from the basal cells through the gaps. The pseudopodia were frequently related to peripheral cytoplasmic microfilaments. Quantitative data confirmed the progressive nature of this loss, with values for SSLD,BM being of the order of 98 per cent., 88 per cent., 76 per cent, and 42 per cent. for normal epithelium and for the hyperplastic, dysplastic and carcinomatous lesions respectively. The loss of lamina densa is discussed in relation to the specificity of the response and to the development of features indicative of motility in transforming cells.

Quantitative studies of hemidesmosomes during progressive DMBA carcinogenesis in hamster cheek-pouch mucosa.

The present study was designed to establish whether there are changes in hemidesmosomal distribution during defined stages of chemical carcinogenesis in hamster cheek-pouch epithelium. 0.5% DMBA in liquid paraffin was applied thrice weekly to hamster pouches and tissue samples were obtained at regular intervals and assigned to hyperplastic, dysplastic and carcinomatous groups on the basis of histological criteria. Untreated pouches served as controls. Following a strict sampling regime, electron micrographs were obtained from the epithelial-connective tissue junction and, using stereological intersection counting, the relative surface area of basal plasma membrane (BPM) occupied by hemidesmosomes was estimated. In normal epithelium 40% of the BPM is occupied by hemidesmosomes. During carcinogenesis, values decrease progressively and significantly to 35% in hyperplasia, 28% in dysplasia and 13% in carcinoma. A decrease in the relative area of hemidesmosomes would therefore appear to contribute to the increased motility of epithelial cells during a connective-tissue invasion and cellular metastasis.

A Relatively Inexpensive Microprocessor-Linked Digital Plamimeter for Electron Microscopic Morphometry

Stereology provides a theoretical basis for powerful morphometric methods for the estimation of three-dimensional structural parameters from two-dimensional electron micrographs of cells and tissues. These methods assume at the start that one has a sufficiently large set of micrographs containing valid structural data. The task of obtaining from these micrographs the large quantity of data needed to get statistically valid results has been eased in two general ways. Sampling of data in the micrograph can be done rapidly by point and intersection counting methods. An alternate method, planimetry, obtains all the data in the micrograph, but in general is more time-consuming than point and intersection counting. Some of the relative inefficiency of planimetry is compensated when a digital planimeter is coupled with a computer. Areas and lengths can be computed simultaneously as fast as profiles are traced. Furthermore, rapid and numerically accurate compilation and statistical analysis of the data can be done automatically as the planimetry is done, not as a separate step after the data have been obtained.