Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

Morphometry of transport tissues in a freshwater crustacean

Gills and fenestra dorsalis of the syncarid crustacean Allanaspides helonomus were examined with the electron microscope. Their epithelia were characterized using morphometric measurement, with a view to assessing potential for ion transport and respiratory activity. The results obtained show distinctions between the paired gills and allow the possibility that one gill of each pair is respiratory and the other osmoregulatory. The fenestra dorsalis has been tentatively assigned a transport function, although the nature of its transport load remains uncertain. The detailed morphometric analysis provided useful insights into the nature of the tissues studied.     

Morphological characteristics of the endocrine pancreas in alloxan diabetes after cyclosporin A administration.

Histological, immunocytochemical, morphometric and electron microscope studies were carried out on the pancreas of alloxan diabetic rats pretreated with cyclosporin A. High mortality, severe destruction of pancreatic B-cells and presence of sporadic mononuclear infiltrations in islets and around excretory ducts were observed. The results obtained show that cyclosporin A potentiates the toxic effect of alloxan on the pancreatic B-cells.     

The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used

Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.     

Accumulation of myocardial lipid droplets in dexamethasone-treated mice

Summary Conventionally fixed and plastic-embedded myocardial tissue from mice treated with 1 mg/kg body weight dexamethasone for 48 h was examined in the electron microscope. The dexamethasone-treated mice showed a marked increase in the accumulation of lipid droplets as compared with control animals. The per cent volume of lipid droplets, calculated by morphometric analysis, showed a significant increase in the dexamethasone-treated mice. No other ultrastructural difference between dexamethasone-treated mice and controls was observed.     

Morphometric and three-dimensional study of platelets during activation in the rat

In vivo activation of platelets, produced by damaging an artery in the rat with ultrasound, was studied with the electron microscope. We performed both a three-dimensional reconstruction by thin serial sections and a morphometric study of the activation process. This is characterized by exocytosis of the content of granules, widening of tubules of the open canalicular system, and emission of pseudopodia. The three-dimensional reconstruction suggested that some extended pseudopodia adhere to the arterial intima, and confirmed former observations that a locomotor apparatus differentiates in platelets adhering to the arterial intima. We speculate that the contraction of some pseudopodia may pull the platelet body toward the arterial wall. The morphometric study revealed that during activation the volume of the open canalicular system volume increases and that of the dense granules decreases; all other compartments did not change.     

Leucine inhibition of autophagic vacuole formation in isolated rat hepatocytes

An electron microscopic, morphometric analysis of isolated rat hepatocytes revealed a 70% decrease in the early forms of autophagic vacuoles after administration of leucine. The lysosomal degradation of protein was reduced by only about 30% under the same conditions. These observations suggest that leucine is a major regulator of the bulk autophagy observable in the electron microscope, but that this type of autophagy contributes only about one-half of the total amount of protein degraded in lysosomes. Asparagine inhibited lysosomal protein degradation more strongly than did leucine, but had no significant effect on the amount of autophagic vacuoles. Leucine and asparagine would therefore seem to exert their effects on lysosomal protein degradation through different mechanisms.     

Measurement of number and cross-sectional area of basal cell pseudopodia: a new morphometric method

A method of morphometric quantitative of the number of pseudopodia per individual basal cell and the ratio of the total cross-sectional area of the pseudopodia to the base area of the basal cell, using the transmission electron microscope, was developed. The diameters and areas of the bases of basal cells and the pseudopodia were also obtained. The number of pseudopodia per basal cell (N) and the ratio of the areas (F) measured in normal human uterine cervical epithelium were 34.22 and 0.338, respectively. The values observed in reactive atypia were 23.62 and 0.188; and those in mild dysplasia of the cervical epithelium (the earliest premalignant condition of the cervical epithelium), 26.98 and 0.226. There were statistically significant reductions in the number of pseudopodia per cell (N) and the ratio of areas (F) in the latter two pathological conditions compared to the controls. This morphometric method provides higher sensitive means by which one can quantify the characteristics of pseudopodia in various premalignant epithelia.     

Marginal and folliculo-stellate cells of the pituitary gland of the rat. A comparative morphometric study in lactating animals

We examined non-granulated pituitary cells (folliculo-stellate cells and anterior and posterior marginal cells of the pituitary cleft) in lactating and virgin female rats by means of electron microscopy and morphometry. Ultrastructure and morphometric parameters of the cells lining the pituitary cleft were similar in the two groups of animals. On the contrary, folliculo-stellate cells showed a marked nucleocytoplasmic activation in lactating rats in the electron microscope, which was confirmed by morphometric measurements. These results are not consistent with the hypothesis that the three cell types share the same reactivity during pituitary hyperfunction and that they have a common function, as suggested by purely morphological studies in various endocrine conditions. We believe that quantitation of cell response during such stimuli could be useful to further elucidate this matter.     

Geometrical characteristics of mitochondria and active presynaptic zones in the dorsal horn of the cat spinal cord

A morphometric electron microscope study was carried out on the ultrastructure of 140 presynaptic terminals (PT) in the dorsal horn of the cat spinal cord. Some spatial characteristics of mitochondria and active zones (AZ) for these PT were examined using statistical stereological analysis techniques. The distribution of 3-dimensional mitochondrial radii was determined, together with average mitochondrial volume, mean area of the external membrane, and average numbers of the test population falling within the PT. Distribution of diameters and estimates of mean area of AZ were obtained, as well as of mean value of synaptic clefts. The relationship between findings from morphometric research and parameters of processes underlying ionic transmembrane diffusion and accumulation is discussed.Dnepopetrovsk State University. A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 741–747, November–December, 1989.     

Effects of met-enkephalin on the adrenocorticotropic cells of the rat pituitary

The effects of chronic administration of met-enkephalin (40 micrograms/d during 20 days) on ACTH producing cells of the rat adenohypophysis have been studied both in light (PAP-immunocytochemistry for ACTH) and electron microscope. In addition a morphometric analysis and a percent distribution of secretory granules were performed. The ACTH cells of treated animals showed ultrastructural signs of hyperactivity, and some of them vacuolization (small vacuoles or a large central vacuole). The secretory granules of the experimental animals are larger and more spherical than the ones in the untreated and the control animals. We discuss the possible mechanism of action whereby met-enkephalin influences ACTH cells.     

Aggregation of Escherichia coli B/r A during agar filtration: effect on morphometric measurements.

We have investigated the phenomenon of particle aggregation in a sample of 71,038 Escherichia coli B/r A cells in balanced exponential growth, during preparation for electron microscopy by agar filtration. The bacteria were photographed in a transmission electron microscope and the dimensions and spatial relationships among all the members of each aggregate were recorded using an interactive image processing system. The proportion of aggregated cells, 22%, is much greater than that found by direct count in a light microscope (7%), implying that most aggregation takes place during the preparation stages. The aggregated cells are about 1% narrower than the free cells, because of mutual compression, and 1.5% longer, because of a selection bias in favor of longer cells. From a statistical analysis of the data, we conclude that the clustering of cells into aggregates in the course of sample preparation is the result of random encounters during the settling on the collodion membrane and of the changing surface tension during the drying process. A method is proposed to correct morphometric measurements for the distortion caused by cellular aggregation of this kind.     

Comparative egg morphology of six Meccus species and Triatoma recurva (Stål, 1868) Hemiptera: Reduviidae

We investigated the morphology and morphometry of eggs from the colonies of the Entomology Laboratory at ENCB‐IPN belonging to six species of the genus Meccus and Triatoma recurva, using both light and scanning electron microscopy. Egg ornamentation is an important parameter to consider in the differentiation of species. Samples were observed under a scanning electron microscope. Measurements of fifty eggs per species included length, width, and opercular diameter, which were used for the morphometric analysis. The results showed that the seven species display a polygonal ornamentation dominated by hexagons; the operculum shows also a polygonal ornamental characteristic in each species. Morphometry provided valuable information for discriminating between closely related species of the genus Meccus and Triatoma recurva, a species akin to this group, thereby facilitating the complete discrimination of these species.     

A scanning and transmission electron microscopic study of the bat lung

The lungs of two adult species of bat Epomophorus wahlbergi and Miniopterus minor fixed with 2.3% glutaraldehyde were processed for SEM (scanning electron microscope) and TEM (transmission electron microscope) examination by the standard procedures. The bat lung comprised a blood and air conducting zone (consisting of bronchi, bronchioles and large blood vessels), the intermediate zone (made up of alveolar ducts), and the respiratory zone, which consisted of alveoli and blood capillaries. The interalveolar septa comprised basically granular pneumocytes (type II cells), squamous pneumocytes (type I cells), endothelial cells, and, in the interstitium, collagen and elastic fibres with occasional fibrocytes. Blood capillaries were interposed in the interalveolar septa, thus bulging into adjacent alveoli. It was noted that grossly, architecturally and structurally, the bat lung was similar to that of a terrestrial mammal. However, in previous morphometric and physiological studies it has been found that bats have a large lung, a thin pulmonary blood-gas barrier, a large pulmonary capillary blood volume, and high haematocrit and haemoglobin concentration. The bat lung, while retaining the basic mammalian pulmonary design, is well adapted to provide the large amount of oxygen demanded by flight. The avian pulmonary design (the lung-air sac system) is thus not a prerequisite to flight.     

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

Summary The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.     

Circadian changes in prolactin cell activity in the pituitary of the teleost Poecilia latipinna in freshwater

Summary Quantitative morphometric studies with the electron microscope were made on the prolactin cells of wild freshwater sailfin mollies taken in Florida in August at four different times of day. The results indicate a circadian rhythm in the prolactin cell, the period of highest synthetic activity being from midday to evening, as indicated by the condition of nucleus, Golgi apparatus and rough endoplasmic reticulum, and by the incidence of granule release profiles. No circadian changes were detected in the ACTH cells.Several distinct sites of prolactin granule release were recognised. However, there was no conclusive evidence of granule release by any mechanism other than classical exocytosis.We thank Mr. W. Thomson for technical assistance and Ann Grier for help in collecting the fish. T.F.C. Batten is in receipt of an S.R.C. Research Studentship.     

Performance of a TV image analysis system as a microdensitometer

The performance of a TV image analysis system combined with an automated microscope (the Leitz TAS plus) as a microdensitometer and morphometric device was investigated. There was a strict linear correlation (r greater than .99) between the physically defined optical transmission values and the resulting electronic signals from the plumbicon TV camera for the whole area displayed on the monitor. The shading of the optoelectronic system had a coefficient of variation (CV) of 1.42% for measurements in the center of the displayed area, but a CV of 3.55% for measurements over the whole monitor area. Densitometric measurements remained stable 15 minutes after putting the microscope lamp into operation (T less than 0.075%, remeasuring every two minutes). The geometric distortion, measured as different ferret diameters of ideally round latex particles, ranged from +/- 0.5% to +/- 1.0% deviation over the entire displayed area. These results indicate that densitometric and morphometric measurements with this equipment are sufficiently precise and reproducible when performed in the center of the area displayed on the monitor.