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Three-dimensional morphometry in scanning electron microscopy: a technique for accurate dimensional and angular measurements of microstructures using stereopaired digitized images and digital image analysis.

A method for accurate dimensional and angular measurements of microstructures analysed in the scanning electron microscope is described. The method considers central and parallel projections and involves (a) digital image acquisition of stereopaired images from the scanning electron microscope's photodisplay, (b) generation of 3D-image representations, (c) setting of measuring points in the digitized stereopaired images, (d) computation of exact space coordinates (x/y/z) from the corresponding point coordinates (xL/yL; xR/yR), (e) determination of distances and angles between consecutive corresponding points using vector equations, and (f) transfer of computed data into spreadsheets of the data analysis software using dynamic data exchange with simultaneous graphical display of the frequency distribution of variables. Measurements performed on specimens with known dimensions (grid with 10 microm wide square meshes, polystyrene beads with 0.33 microm diameter) and angles (synthetic crystals of K(Al,Cr)[SO4], CuSO4.5H2O and NaCl) revealed a high accuracy in dimensional as well as angular measurements (total error 1 +/- 0.5%). In Monte Carlo experiments the overall error was found to depend strongly on the size of the measured structure relative to the size of the measurement field (field width).

Image Processing, Computer-Assisted↗

Study of Ligmed-A, an antidiarrheal drug based on lignin, on rat small intestine enzyme activity and morphometry.

This study aimed to determine the potential toxic effect of 4-day oral treatment with a lignin-based formulation on the enzymatic activity and morphology of the small intestine of rat. Ligmed-A is collected from sugarcane and is used to treat diarrhea in weaning pigs. The compound is about 90% lignin, an insoluble polyphenolic constituent of plants and a component of dietary fiber. The duodenal, jejunal and ileal mucosa of control rats and those receiving 2 g/kg Ligmed-A showed similar protein contents of about 100 mg/g. The sucrase and alkaline phosphatase activities of the three intestinal segments were unaltered after administration of the compound. Nonhistological alterations were observed after treatment. Our results, together with those of previous studies which found no toxicological effects, indicate that Ligmed-A could provide a potent antidiarrheal treatment in the veterinary area. However, further studies are required to examine its use in humans.

Administration, Oral↗

Prognostic implications of microvessel morphometry in diffuse astrocytic neoplasms.

Astrocytic brain tumours, particularly malignant astrocytomas, are recognized to be highly vascular neoplasms with potent angiogenic activity. Recent research has shown that quantification of microvessel density (MVD), as a measure of the degree of angiogenesis, constitutes a strong prognostic indicator in patients with astrocytomas. However, the significance of other morphometric aspects of microvessel network has not been tested so far. In this report, histological sections from 70 astrocytomas (grades II to IV), immunostained for CD34, were evaluated by image analysis for the quantification of MVD, total vascular area (TVA), and microvascular branching, as well as several morphometric parameters related to vessel size or shape. Minor axis length increased with grade (P = 0.045) but MVD and TVA presented a peak in grade III (P = 0.033 and P < 0.001, respectively). Size and shape related parameters affected survival in univariate analysis of grade IV and grades II/III, respectively. In multivariate analysis, only branching counts, along with age and grade, were the independent predictors of survival. Although MVD, TVA and branching counts were adversely related to disease-free survival in grades II and III (univariate analysis), only TVA remained statistically significant in multivariate analysis. It is concluded that TVA and branching counts are prognostically more informative than MVD for patients with diffuse astrocytic tumours.

Adolescent↗

Red muscle development of gilthead sea bream Sparus aurata (L.): structural and ultrastructural morphometry.

The transverse red and white muscle area, the superficial red muscle fibres area and their percentages of mitochondria (%mit), myofibrils (%myof) and sarcoplasm (%sarc) were determined in the Mediterranean teleost gilthead sea bream, Sparus aurata (L.). Fish aged from hatching to 78 days were studied. The proportional growth of the red and white muscles was higher for the red muscle in the first half of the larval stage (1-35 days). Then the opposite relationship was observed. The hypertrophic growth of the superficial muscle fibres was continuous except in the first week after hatching. The percentage of mitochondria and percentage of myofibrils showed a significant change just after the half of the larval stage. Whereas the %mit of the superficial muscle fibres was higher than %myof from the first week after hatching to 35 days (average 66.64%), then the %mit decreased significantly and at 73-78 days both parameters were close to an average value of 50%. The meaning of these morphological changes is discussed in relation to the functional role of the red muscle of larvae and the onset of the gills respiration.

Aging↗

Morphometry and histochemistry of the semitendinosus muscle of tufted capuchin monkeys (Cebus apella Linnaeus, 1758).

Considering monkeys are animals closely related to the human, and semitendinosus muscle has been used in many postural research experiments, we have decided to study its histochemical characteristics. Samples were removed from the proximal, middle and distal regions of the semitendinosus muscles of five adult male tufted capuchin monkeys and observed for reaction with m-ATPase (with alkaline and acid pre-incubation), nicotinamide adenine dinucleotide tetrazolium reductase, and haematoxylin and eosin. The muscle fibres were classified as fast glycolytic (FG), fast oxidative glycolytic (FOG) and slow oxidative (SO) and quantified in terms of frequency and area. The three regions of the semitendinosus muscle showed no significant differences in frequency or area of the respective fibre types, and therefore the muscle can be considered histoenzymologically homogeneous. FG fibres presented higher frequencies and larger areas. The sum of FOG and SO fibres was 57.5%, suggesting that the semitendinosus muscle of the tufted capuchin monkey is adapted to an action involved in posture maintenance and in long arboreal dislocation. The present data agrees with the notion of differentiated quadrupedalism in some primates, which support a lower percentage of their weight on the fore limbs.

Analysis of Variance↗

Accuracy of body weight prediction in Alpine ibex (Capra ibex, L. 1758) using morphometry.

The relationship between weight and body measurements of 227 Alpine ibex Capra ibex (162 males and 65 females, age range: 2-16 years) were analysed by linear and multiple regression. The animals were live-captured across all seasons of the year, in the Western Italian Alps, 1986-1994. The relationship between weight and body measurements changed with sex and season. In male ibex, the best annual estimation of the body weight was obtained using as predictor variables the squared value of the chest girth and age. In females, the best predictive variables were age, total body length and withers height. Age was always selected as a predictor variable, when considered in the prediction models. The use of chest girth and the squared value of the chest girth as predictive variables in the regression equation, being the most highly correlated linear measurements with total weight, did not result in an accurate prediction of ibex body weight. The best prediction was obtained using age and different linear measurements in stepwise multiple regression, nevertheless the standard error of the estimate and the magnitude of the 95% prediction interval were great. The use of linear measurements cannot be considered accurate nor precise predictors of body weight in Alpine ibex.

Animals↗

Age-related morphometry of equine incisors.

In the present study the age-related morphological characteristics of 948 equine incisors were investigated. After extraction, total incisival length and root length were measured at the vestibular side of the teeth. Equine incisors reach their maximal length 2-3 years after eruption. Notwithstanding severe occlusal wear, this maximal length is maintained during most of the horses' life due to prolonged root formation. Root formation, at the rate of 2.5 mm per year, starts at the age of 5-6 years and continues until the age of 17. As the root of the incisor develops, its apical foramen narrows and changes position. In young horses the apical foramen is situated at the apex of the tooth, whereas in older individuals it is located at the mesial, distal, or lingual side of the tooth at a distance of 5-15 mm from the dental apex. In horses aged over 20 years apical foramina are still present. Radiographic imaging is a good method with which to obtain reliable information concerning the total incisival length and the size and position of the apical foramen.

Aging↗

Evaluation of the growth dynamics of Trichophyton rubrum cultures by morphometry and non-linear curve fitting analysis.

To determine the size of dermatophyte thalli, a point-counting procedure with a square lattice grid is proposed instead of using the one-dimensional diameter as the size parameter. For an analysis of the growth dynamics of such colonies, the area values might be subjected to a non-linear curve-fitting procedure. To demonstrate the method, samples of 15 Trichophyton rubrum strains, isolated from human skin and cultured concomitantly on Sabouraud and Selective agar during a period of 8 weeks, were used. The morphometric assessment of the area of the thalli, subjected to non-linear curve fitting, disclosed only a slight difference in the growth dynamics.

Dermatomycoses↗

Influence of sample preparation, staining procedure and analysis conditions on bull sperm head morphometry using the morphology analyser integrated visual optical system.

The importance of standardizing the procedures of sample and slide preparation for computer-assisted morphologic analysis has been emphasized in human and veterinary andrology. The purpose of this study was to optimize slide preparation (dilution grade and sperm washing), staining procedures and analysis conditions (colour of light source and objective magnification) for the morphometric analysis of bull spermatozoa using the Hamilton Thorne morphology analyzer integrated visual optical system (IVOS). For experiment 1, one ejaculate was collected from one bull and diluted to 200,000-300,000 spermatozoa/microl. Slides were prepared and stained using seven different procedures: rapid Papanicolaou (PAP), rapid Papanicolaou with prolonged staining times (PAP+). Diff-Quik (DIF), haematoxylin (HEM). Farelly (FAR), Spermac (SPER) and the modified GZIN (MGZIN) staining. All slides were analysed using a Hamilton Thorne Morphology Analyser IVOS equipped alternatively with a red, green or blue light source, and a 40x or 100x oil immersion objective. Recognition and digitization errors as well as morphometric parameters were determined. The IVOS was unable to detect DIF-stained spermatozoa. The GZIN and the SPER staining as well as the blue light source led to unsatisfactory results. Among the staining methods examined, the FAR, HEM, PAP+, and PAP staining, preferably in combination with the green light source, and the 40x objective yielded optimal results concerning sperm recognition and digitization. The 100x objective did not allow reliable analysis of the sperm heads because of a frequently appearing digitization error. For experiment 2, three ejaculates were collected from each of three bulls and diluted to five dilution grades (100 000-500 000 spermatozoa/microl). An aliquot of each dilution grade was washed additionally. The percentage of correctly digitized sperm heads decreased with increasing spermatozoal concentration. However, the evaluation speed increased. The range of 200 000-300 000 spermatozoa/microl appeared to be a reasonable compromise for both criteria. Sperm washing failed to further improve the analysis results. Sperm head dimensions were influenced significantly by all variations of the methods in both experiments. In conclusion, using the proposed methods, the IVOS allows precise and reliable morphometric analyses of bull spermatozoa. The consistent application of these procedures may lead to an inter-laboratory standardization and to further establishment of generally accepted morphometric criteria used in human andrology (e.g. World Health Organisation or strict criteria).

Animals↗

Gross morphometry of the bovine placentome during gestation.

Forty-seven pregnant uteri were collected from an abattoir at eight chronological stages of gestation. Assessments were made of placentome number, weight, length shape and ratio of foetal to maternal tissue by weight. The study showed that there was a great variation in number, size, weight and shape of placentomes. There was no significant change in the total number of placentomes during gestation. Both mean weight and length increased significantly during gestation, but there was a significant change in the relationship between the two as gestation progressed. There was no correlation between the number of placentomes and the mean weight or length of the placentomes. The development of the placenta in the non-pregnant horn was significantly different from that of the pregnant horn, with fewer, smaller, lighter placentomes. However, there was no evidence that placentome development in the non-pregnant horn was affected by placentome development in the pregnant horn. The proportion of the placentome that was maternal tissue increased during gestation, but there was no significant effect of placentome number, or individual placentome weight or length on this proportion. Two placentome shapes were identified, but no effect on foeto-maternal proportion or relationship between shape and total placentome number or weight was identified.

Abattoirs↗

Evaluation of pulmonary volumetric morphometry at the light and electron microscopy level in several species of passerine birds.

The lungs of 3 small passerine species, having similar body mass but different diurnal activity patterns, were analysed morphometrically to assess the relationship between diurnal activity and pulmonary volumetry at the light and electron microscope levels. The percentage volumes of the major lung and exchange tissue components of the 3 species--an aerial insectivore, a foliage gleaner/nectarivore and a ground forager--were strikingly similar, and consistent with literature values for other passerine species. The only significant difference found was exchange tissue plasma volume and pulmonary haematocrit, with the ground-foraging, low activity Malurus splendens having significantly lower values than the other 2 species. This may indicate that cardiovascular parameters are more important determinants of metabolic activity in small passerines than aspects of pulmonary anatomy.

Animals↗

Prognostic importance of the nuclear proteins p53 and Rb in conjunction with DNA, nuclear morphometry and grading in endometrial carcinoma.

In a series of 227 cases of endometrial carcinomas in FIGO stages I-IV, treated during the years 1984-89, immunohistochemical staining for the protein products of the two tumor suppressor genes p53 and retinoblastoma (Rb) were evaluated as prognostic factors with regard to tumor stage, FIGO grade, nuclear grade, morphometric nuclear parameters, DNA ploidy and S-phase fraction. Long-term survival analyses were endpoints and the Cox multivariate technique was used to evaluate the prognostic factors. In 20% of the cases p53 was positive. This was a genuine high-risk group associated with primary advanced carcinoma, nonendometrioid histology, poorly differentiated tumors, severe nuclear atypia, DNA aneuploidy, primary persistent tumors, recurrent tumors and a poor long-term survival rate (37% 5-year survival). In patients dying of their disease, 54% of the tumors stained positive for p53, compared with only 10% of the tumors not killing their hosts. Positive p53 staining was more common in older women. A pathologic Rb status (negative staining) was recorded in 6% of the cases. The Rb factor had only a minor influence on long-term survival and was not significant in multivariate analyses. The p53 staining status was the second most important prognostic factor after the nuclear grade in Cox multivariate analyses, after correcting for stage and age. Immunohistochemical staining for p53 protein should be included among previous available and important prognostic factors in endometrial carcinoma.

Journal Article↗

[Conventional and analytic morphometry using the IBAS-1 computer in experimental Trypanosoma b. brucei infection].

A comparative morphometrical study of the experimental infection with T. b. brucei was realized using the conventional method and the IBAS-1 analytic computer. The studied strain of T. b. brucei was preserved during 10 years in laboratory conditions, through inoculations from mice to mice. The effect of a low dose of suramin upon the percentage of different morphological forms of the parasite was analysed and compared with the untreated lots. Certain differences could be appreciated in the results of the two methods of morphometrical studies. The presence of posteronuclear, multinuclear and "stumpy" dividing forms was confirmed in our study. The administration of low doses of suramin produces a serious increase of the number of the dividing, multinuclear and "stumpy" forms.

Animals↗

Relationships between Leydig cell morphometry and plasma testosterone during postnatal development of the monkey, Macaca fascicularis.

In neonates (0 to 3-4 months), the testis contained a mean number of 4.6 X 10(6) Leydig cells representing 4.2 % of its volume; Leydig cell cytoplasm contained 10.2 % of SER. In infants (up to 45 months), Leydig cells regressed but their number increased; their volume density did not change. Leydig cell cytoplasmic volume (454 microns3 ), which was about 2.5-fold less than in neonates (1 119 microns3 ) or adults (1 170 microns3 ), contained only 8.7% of SER. During meiosis stage (38-52 months). Leydig cell numbers and volume density did not vary but the cells reached a maximal size and an amount of SER comparable with that at birth was measured. When spermatogenesis was complete, the Leydig cells represented no more than 0.8% of testis volume, but their number and SER content were significantly increased. Except for a significant decrease when spermatogenesis was completed, Leydig cell lipid content did not change during development, and the volume density of mitochondria did not vary. The mean level of plasma testosterone was 2 ng/ml in neonates and 0.4 ng/ml in infants; it increased to 3 ng/ml during onset of meiosis and reached 10 ng/ml in adults. The profile of testosterone was positively and significantly correlated with the total volume and total number of Leydig cells (P less than 0.01 and P less than 0.02, respectively) and with changes in their cytoplasmic volume (P less than 0.001). Moreover, plasma testosterone levels were positively and significantly correlated with changes in Leydig cell SER content i.e. SER volume density and mean absolute volume per cell (P less than 0.001), total SER in the whole testis (P less than 0.01).

Aging↗

Morphometry of the heart of budgerigars (Melopsittacus undulatus), Alisterus parrots (Alisterus s scapularis) and common buzzards (Buteo buteo).

In order to establish data and reference values for the thickness of the myocardium and the length of the left and the right ventricle of the avian heart, the hearts of 14 budgerigars (Melopsittacus undulatus), five Alisterus parrots (Alisterus s scapularis) (also known as Australian king parrot) and 10 common buzzards (Buteo buteo) of both sexes were examined according to a standard protocol. In order to compare the results of birds of different sizes all data were related to the size of the bird's body (length of the sternum) and the results of the measurements of the myocardial thickness in relation to the length of the heart. Results of different zones were compared by means of statistical methods within one species. Besides that a comparison between the different species was performed. Only minor significant differences were noted.

Animals↗