A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.     

Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study

The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.     

Confocal stereology and image analysis: methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images

A short review of confocal stereology and three-dimensional image analysis is presented, pointing out the achievements accomplished in this field by the Department of Biomathematics (Institute of Physiology, Prague). One of the methods of confocal stereology, the fakir method for surface area estimation, developed by this laboratory, is described. Methods for automatic measurement of geometrical characteristics of microscopical structures, based on 3-D image processing or surface triangulation, are discussed and compared with interactive stereological methods. Three-dimensional reconstruction programs and software implementation of stereological and digital methods as well as their practical applications are presented. The future trends are discussed.     

Stereological analysis of phagocytosing cells

Various complexes of phagocytes with adsorbed (I) and entrapped (J) particles are formed during phagocytosis. Method of stereological reconstruction is proposed that allows to demonstrate the actual distribution of these complexes on the basis of morphometric analysis of their ultrathin sections. The principle of the method lies on the probability simulation of section distribution for a given distribution of complexes and on the solution of the reverse problem by stepwise determination of the relative quantity of each complex type (from the most complicated to the most simple one, when I = 0 and J = 0). The stereological analysis of phagocytosing murine peritoneal macrophages revealed an absolutely different and more adequate kinetical picture of phagocytosis, as compared to the morphometric data.     

Method for the calculation of the 50% effective dose of biologically active agents

A newly proposed method for the mathematical and graphic determination and calculation of ED50 (LD50) on the abscissa at the meeting point of the cumulate of dead and the cumulate of survived animals (in absolute figures) in "dose-effect) experiments is described. "The method of meeting cumulates) for the calculation of ED50 (LD50) is simple, eliminates unnecessary calculations, yields results, highly similar (95-100%) to those obtained by other methods and may be used in different medico-biological studies.     

Structural and functional analysis of the respiratory burst in the phagocytosing macrophage

The interrelation between structural changes and oxygen consumption by the phagocyting macrophage was studied. The mean number of phagocyted particles was estimated by the method of stereological transformation. It is found that the uptake of yeast particles and CN- -nonsensitive oxygen consumption is related to the concentration of yeast cells in the incubation medium. A positive correlation was established between the oxygen consumption and the mean number of phagocyted particles. The results obtained may suggest that the "respiration burst" takes place in the contact area of the macrophage and the phagocyted material, and its extent probably depends on the surface of that contact area.     

Photoreceptor number and outer segment disk membrane surface area in the retina of the rat: stereological data for whole organ and average photoreceptor cell

A random sampling scheme is employed to obtain stereological estimates of disk membrane surface area in the entire retina and in the average photoreceptor cell. The scheme involves the use of vertical sections with combined light and electron microscopy at several magnification levels. Left and right retinas from six albino animals were analysed. There were no significant lateral differences. On average, the retina had a volume of 16 mm3, thickness of 200 μm and surface area of 80 mm2 (representing about 56% of the external surface of the eyeball). Photoreceptor disk membranes within outer segments amplified total retinal surface by almost 1000-fold (final surface 770 cm2 per retina). The retina contained 3×107 photoreceptors (packing density 374 000 mm-2) with an average disk membrane surface area of 2600 μm2. Mean nuclear volume in photoreceptor cells was 59 μm3 and the coefficient of variation for the distribution of nuclear volumes was 57%. The data are consistent with an average of 700 disks per photoreceptor cell, a membrane area of 4 μm2 per disk and a convergence ratio of ～260 photoreceptors per optic nerve fibre. The basic scheme could be modified for other species and for direct cell counts conducted on rods and cones separately.     

Functional assessment of pulmonary capillary surface area in the 2-mo-old lamb.

We recently reported that measurements of the maximal velocity of pulmonary endothelial angiotensin-converting enzyme (Vmax) in vivo provide information regarding microvascular surface area in the developing lamb. To obviate any subtle influences of development on Vmax aside from simple increases in surface area, we correlated Vmax with postmortem stereological assessments of alveolar surface area in the relatively mature lung of the 2-mo-old lamb (n = 14). We attempted to increase the range of surface area beyond its normal variability by injecting nine of the lambs with bleomycin, an antineoplastic agent with significant pulmonary toxicity in other species. Vmax, measured shortly after birth and then weekly, increased monotonically in all lambs. Despite their wide dispersion, Vmax and the stereological determinations correlated strongly at 2 mo of age, confirming that Vmax is a robust indicator of the surface area of the air-blood barrier. There was no significant difference in either measurement between the control lambs and those treated with bleomycin, suggesting that the newborn lamb is resistant to the effect of this agent.     

Electroplethysmographic determination of the regional relative engorgement of the lungs

On the strength of the theoretical research of a biophysical model of the lung structure and the following experimental checking of the results obtained a method of calculation of the regional relative blood volume in the lungs by the data of electroplethysmographic examination of the lungs and measurement of specific electrical conductivity of the blood is proposed. Calculation formulae for the determination of the regional relative blood volume in the lungs were derived their working ranges within the limits of application of the proposed calculation method in clinical and physiological examination of the lungs were established.     

Structural and functional study of acidification in the process of phagocytosis

Morphometry and cytofluorometry of the kinetics of phagocytosis were applied to study the quantitative mechanisms of acidification in the area of contact between the plasma membrane of macrophages and Candida albicans yeast cells conjugated with fluorescein isothiocyanate. It was found by stereological transformation of the morphometry data that the main part of macrophages phagocytose the limiting amount of particles by minute 5-10. A good agreement was established between the cytofluorometric histograms and the stereological data. This made it possible to evaluate, using the calibration curve, the pH on the surface of the phagocytosed material based on the fluorescence quenching. As an advantage of the suggested comprehensive approach to the study of acidification, the authors stress the possibility of making structural-functional analysis of the rearrangements occurring in the intact phagocytosing cell.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in kappa-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a kappa-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in kappa-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.     

On the possibility of determination of stereological characteristics of interactions of biological micro objects with nanoparticles based on analysis of 2-dimensional images of cytological and histological specimens

In this article the method for studying 3-dimensional (stereological) characteristics of interactions of biological micro objects with nanoparticles on a basis of morphodensitometry analysis of standard 2-dimensional images of cytological and histological specimens is proposed. The performance of the task of determining the distance from registered nanoparticle to the center of the nucleus of the cell is described in detail. It is shown that using specific nanoparticles the results obtained may find application in science and diagnostics. Furthermore, it is possible to employ these results in nanotoxicology, in particular for determination of quantity characteristics of translocation of nanoparticles of different types in biological structures.     

Development of the accessory glands in the genital tract of female Teleogryllus commodus WALKER (Insecta, Orthoptera)

The development of the female accessory glands in Teleogryllus commodus was studied by detailed morphometric and stereological analyses. In addition, a microsurgical method was developed to quantify the gland secretion. The morphometric results yield evidence that the glands are subject to a significant growth during peak differentiation, starting immediately after the adult moult. The gland growth is exclusively caused by a hypertrophy of single gland cells with respective volume gains between 400 and 700%. According to the stereological results, the volume of mitochondria per cell is marked by an up to fourfold increase during peak differentiation. Other cell structures (rER, sER) are characterized by a similar propagation behavior. The nucleus and nucleolus grow simultaneously with the cell, indicating high production of site-specific macromolecules. Infolds of the basal cell membrane cause a progressive enlargement of the basal cell surface ensuring an increased uptake of secretory precursors from the hemolymph. Quantitative studies show that the total production of secretion increases with proceeding age and can be correlated with a rising egg-laying activity, starting on the eighth day of adult's life. This underlines the main function of the secretion as a lubricant for a facilitated transport of the eggs through the ovipositor.     

Stereological Implications of the Geometry of Cones

Given a cone around a curve β in IR³ it is shown that there is no relation between the curvature of β and the volume of the cone but, in contrast to the volume, the surface area of the cone depends, albeit rather weakly, on the curvature of β. The geometric meaning of the stereological formulae when applied to cones is considered and it is noted that stereological methods to estimate the surface area of a cone need to be extremely precise to provide information concerning curvature or tortuosity.     

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

The 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.     

Scale-independent roughness value of cell membranes studied by means of AFM technique

The roughness of cell membrane is a very interesting indicator of cell's health state. Atomic Force Microscopy allows us to investigate the roughness of cell membrane in great detail, but the obtained roughness value is scale-dependent, i.e. it strongly depends on measurement parameters, as scanning area and step size. The scale-dependence of the roughness value can be reduced by means of data filtration techniques, that are not standardized at nanometric scale, especially as far as biological data are concerned. In this work, a new method, based on the changes of values of some roughness parameter (root mean square roughness and skewness) as a function of filtration frequencies, has been implemented to optimize data filtering procedure in the calculation of cell membrane roughness. In this way, a root mean square roughness value independent of cell shape, membrane micro-irregularities and measurement parameters can be obtained. Moreover, different filtration frequencies selected with this method allow us to discriminate different surface regimes (nominal form, waviness and roughness) belonging to the raw cell profile, each one related to different features of the cell surface.     

Integrated stereological and biochemical studies on hepatocytic membranes. III. Relative surface of endoplasmic reticulum membranes in microsomal fractions estimated on freeze-fracture preparations

New methods are required for identifying membranes in subcellular fractions with respect to their origin, if such preparations are to be evaluated morphometrically. One method is freeze-fracturing which reveals intramembrane particles whose size, pattern, and numerical density differ for various membrane types. The question is examined whether the differences in numerical particle density per square micrometer of membrane (alpha) can be used to differentiate membrane vesicles found in microsomal fractions from liver cells with respect to their origin in the hepatocytes. It is found that the range of alpha for the protoplasmic face (PF) of endoplasmic reticulum (ER) membrane (1,900 less than alpha less than 3,250) is intermediate between those for plasma and mitochondrial membranes. Since PF(ER) should appear in the outer leaflet of microsomal vesicles, alpha was estimated on concave profiles of freeze-fracture preparations; the numerical frequency distribution of vesicles with respect to alpha was trimodal, with a major peak around 2,900/micrometer2 and 66% of the vesicles in the range determined for PF(ER). Using a new stereological method, it was calculated that 63% of the membrane surface in these microsomal fractions was of ER origin by this criterion. On the same preparations, an attempt was made to label the ER-derived membranes cytochemically for glucose-6-phosphatase. A line intersection count revealed 62% of the membrane surface to be of ER origin on the basis of marker enzyme labeling. These findings indicate a smaller part of ER membranes in microsomal fractions than would be predicted from biochemical data (77%). The possible reasons for such discrepancies are discussed; shifts in particle densities due to the preparation procedure could lead to an underestimate by freeze-fracturing, whereas the prediction from biochemical data could be overestimates if marker enzymes were not homogeneously distributed.     

Calculation of usable stock

A calculation method is substantiated giving a possibility for the estimation of the population stock per water region covered by a multitude of samples. This task is solved in two stages: (1) construction of surface describing spatial distribution of abundance in samples and (2) numerical integration of volume covered by this surface. Twelve methods of calculation of such surfaces are compared. Criteria are discussed which allow for the possibility to top select the method most suitable for a certain material (filtration, transformation, blanking of sites, scaling, calculation of areas and volumes). Generalization of the Aksyutina area method is suggested for the case when the population is distributed over a large depth range. Examples of calculation of the stock are given for populations of the Pacific sardine Sardinops melanostictus in the Sea of Japan (a flat area) and the wall-eyed pollack Theragra chalcogramma in the Sea of Okhtosk (a three-dimentional area). The results are compared with calculations by the Aksyutina area method and with assessments of the stock obtained from independent sources.     

Evacuation of pelleted feed and the suitability of titanium(IV) oxide as a feed marker for gut kinetics in Nile tilapia

The present study assessed the suitability of titanium(IV) oxide, TiO₂, as a digesta passage marker in Nile tilapia Oreochromis niloticus and studied the shape of the evacuation curve in this species. In three separate trials, fish were given one dose of either 0·5, 0·25 or 0·1% of their body mass (% BME) of feed marked with 1% TiO₂ or 0·5% BME of the same feed without marker. The fish were serially slaughtered at intervals after feeding and the stomach contents analysed for dry mass and marker content. The data for individual trials were analysed with the linear, square root, surface area and exponential evacuation models and parameter comparisons showed that, although the marker interfered slightly with the evacuation process, true meal size could be predicted more accurately from the marker data. The results of an analysis of the combined data sets suggested that stomach evacuation in this species is dependent more on food particle surface area (surface area model) than on stomach content mass (exponential model) as is generally assumed. On the basis of these results, it was concluded that TiO₂ at an inclusion level of 1% is an acceptable marker for quantifying evacuation with a view to predicting food consumption but should be used with caution in digestibility studies.     

A new method for output factor determination in MLC shaped narrow beams

A new method for the measurement of output factors of narrow beams is presented in this work. By combining a new large area parallel plane ionization chamber (PTW model T34070) with a relative film dosimetry the output factors of small square fields of a 6 MV beam shaped by a MLC were measured. Several detectors (three ionization chambers, two solid state detectors and film) and Monte Carlo simulation were also employed to validate this new methodology and also to determine those detectors more suitable for narrow beam output factor determination.The proposed method for output factor measurement has shown to be in a very good agreement with diamond, diode and Monte Carlo results while it is insensitive to position displacements. Several uncertainties associated to the process of narrow beam dosimetry have also been addressed.