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Analysis of morphological relationship between micro- and macromorphology of Mortierella species using a flow-through chamber coupled with image analysis.

Using a flow-through chamber coupled with image analysis, the morphological parameters of 11 Mortierella species were quantified, and the relationship between micro- and macromorphology was investigated. On potato-dextrose-agar plates, 5 species formed rose petal-like colonies, 3 formed large round colonies, and 3 formed donut-like colonies. By observing micromorphology in a flow-through chamber, fungi were divided into 3 groups, classified according to morphological parameters: (i) a group with a high branch formation rate (q(b): tip/microm/h) and a low tip extension rate (q(tip): microm/tip/h); (ii) a group with a low branch formation rate and a high tip extension rate; and (iii) a group intermediate between the former and the latter groups. In suspension culture, group (i) fungi formed a hyphal bundle with a pulpy pellet-like morphology and a pellet core. In contrast, group (ii) fungi showed an aggregation of hyphae without the pellet core. In a narrow-specific hyphal growth rate (mu(l)) range (0.35-0.45 h(-1)), a higher branch formation rate led to increased hyphal branching, resulting in the formation of a hyphal bundle with a pulpy pellet-like morphology and a pellet core. When the branch formation rate was lower than 2 x 10(-3) tips/microm/h, the mycelia formed less branched but longer hypha. Our study surmises that a micromorphology consisting of a high hyphal growth rate (0.4 h(-1)), low tip extension rate (20 tips/microm/h), and high branch formation rate (8 x 10(-3) tips/microm/h) forms the suitable macromorphology for arachidonic acid production.

Hyphae↗

Image analysis of nuclear DNA content and morphometric characteristics of the tumor cells in human astrocytomas.

In order to estimate the degree of malignancy of astrocytomas, we applied image analysis to study the nuclear DNA content and morphometric parameters of individual tumor cells in surgically resected specimens of 20 patients. There were nine low grade astrocytomas (LGA) and 11 high grade astrocytomas (HGA). Although the mean DNA index did not indicate a significant difference between LGA and HGA, five (45%) of the 11 HGA had an aneuploid DNA distribution pattern. By contrast, all LGA had a diploid pattern. The ratio of the S-G2M phase (% S-G2M) of the HGA (17.5 +/- 12.3%) was significantly higher than that of the LGA (7.0 +/- 4.6%). From the morphometric analysis of the area and shape of the nucleus of tumor cells, we obtained quantitative estimates of the degree of nuclear pleomorphism in astrocytomas. The nuclei of the tumor cells of HGA had larger sizes, increased anisokaryosis and were more irregular. In contrast to flow cytometry, image analysis can be used for quantitative studies of morphological tumor cell characteristics of relevance in the grading of malignancy.

Adult↗

Effect of attachment factors (pili plus Opa) on Neisseria gonorrhoeae invasion of human fallopian tube tissue in vitro: quantitation by computerized image analysis.

Pili (P) and opacity-associated proteins (Opa) facilitate Neisseria gonorrhoeae attachment to human fallopian tube epithelium. Subsequent effects on invasion are unproven. Computerized image analysis was used to study the effects of attachment factors on invasion by comparing a P+Opa+ variant to a P-Opa- variant of strain R10 in the fallopian tube organ culture model. Gonococci in sections of infected fallopian tube tissue were identified with FITC-labelled monoclonal anti-gonococcal antibodies. Nomarski DIC microscopy was used to establish anatomic boundaries that excluded extracellular gonococci from invasion measurements. The area of intracellular fluorescence served as an index of gonococcal invasion. With conservative criteria to exclude extracellular gonococci, the per cent of the intracellular area occupied by fluorescent P+Opa+ gonococci was 18% compared to 4.7% for the P-Opa- variant (P < 0.001). Data suggest that P+Opa+ organisms invaded deeper than P-Opa- microbes over the same time period (P = 0.029). Intra-observer variation in invasion measurements was not significant (P > or = 0.85), and inter-observer correlation was high (correlation coefficient = 0.96). Computerized image analysis is a rapid, reliable means of quantifying gonococcal invasion of fallopian tube epithelium. We conclude that gonococcal attachment factors can facilitate events which enhance gonococcal invasion of fallopian tube epithelium.

Antigens, Bacterial↗

Influence of oxygenator type on the prevalence and extent of microembolic retinal ischemia during cardiopulmonary bypass. Assessment by digital image analysis.

We have previously reported the occurrence of microembolic ischemia in the retina during cardiopulmonary bypass, as revealed by fluorescein angiography. This method has been extended by digital image analysis to include quantification of the extent of retinal ischemia and has been applied to a prospective comparative study of 64 patients undergoing elective coronary operations with either a bubble or a membrane oxygenator. Patients with diabetes or clinically evident cerebrovascular disease were excluded. Bypass procedures were standardized in all cases with pulsatile flow and a 40 microns arterial line filter (Pall EC Plus). Thirty patients had bypass with a bubble oxygenator (Harvey H1700) and 34 patients had bypass with a flat sheet membrane oxygenator (COBE CML). In each case retinal fluorescein angiograms were obtained preoperatively and 5 minutes before the end of bypass and were processed with a digital image analyzer (Context Vision GOP-302). Microembolic perfusion defects were identified by digital subtraction of preoperative and end-bypass angiograms and their total area was computed. Results. In the bubble oxygenator group retinal perfusion defects indicative of microembolism occurred in all 30 (100%) patients. In contrast, over half the patients in the membrane oxygenator group had normal retinal perfusion, and the prevalence of perfusion defects (44%; 70% confidence limits 34% to 54%) was significantly less than in the bubble group (p less than 0.001). In addition, those patients in the membrane group had significantly fewer lesions (median 0; 70% confidence limits 0 to 1) than patients in the bubble group (median 2; 70% confidence limits 2 to 2; p less than 0.001) and also had significantly smaller total areas of retinal ischemia (median 0 mm2; 70% confidence limits 0 to 0.16 mm2) than the bubble group (median 0.22 mm2; 70% confidence limits 0.21 to 0.27 mm2; p less than 0.001). There was no relationship between the extent of retinal ischemia and bypass time, arterial blood gas concentrations, volume of cardiotomy suction or donor blood returned to the pump, or recent medication with aspirin. Inferences. Digital image analysis of retinal fluorescein angiograms may provide a method of quantifying microembolic ischemia in the central nervous system during cardiopulmonary bypass. Flat sheet membrane oxygenation appears to provide significantly better protection against microembolic ischemia than bubble oxygenation.

Blood Gas Analysis↗

Full second-order chromatographic/spectrometric data matrices for automated sample identification and component analysis by non-data-reducing image analysis.

A data analysis method is proposed for identification and for confirmation of classification schemes, based on single- or multiple-wavelength chromatographic profiles. The proposed method works directly on the chromatographic data without data reduction procedures such as peak area or retention index calculation. Chromatographic matrices from analysis of previously identified samples are used for generating a reference chromatogram for each class, and unidentified samples are compared with all reference chromatograms by calculating a resemblance measure for each reference. Once the method is configured, subsequent sample identification is automatic. As an example of a further development, it is shown how the method allows identification of characteristic sample components by local similarity calculations thus finding common components within a given class as well as component differences between classes from the reference chromatograms. This feature is a valuable aid in selecting components for further analysis. The identification method is demonstrated on two data sets: 212 isolates from 41 food-borne Penicillium species and 61 isolates from 6 soil-borne Penicillium species. Both data sets yielded over 90% agreement with accepted classifications. The method is highly accurate and may be used on all sorts of chromatographic profiles. Characteristic component analysis yielded results in good agreement with existing knowledge of characteristic components, but also succeeded in identifying new components as being characteristic.

Chromatography↗

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.

Animals↗

Quantification of chemotactic response of quiescent and proliferating fibroblasts in Boyden chambers by computer-assisted image analysis.

The chemotactic response of human gingival fibroblasts to platelet-derived growth factor (PDGF) was investigated in 48-well modified Boyden chambers. Results were quantified using computer-assisted image analysis of propidium iodidestained cells and were compared with results obtained using the conventional method of quantification by direct counting. Quantification by image analysis was found to be rapid and accurate, and correlated closely with results obtained by direct counting. Bromodeoxyuridine (BrdU)-labeled proliferating cells were double stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antiserum. The cycling cells showed a markedly reduced chemotactic response to PDGF, whereas cells in S-phase of the cell cycle did not show any response. This method of quantification of results of Boyden chamber assays is simple and reliable, and allows the use of double labels to investigate the chemotactic response of subpopulations of cells within a heterogeneous population of cells.

Cell Cycle↗

Carpal bone maturation assessment by image analysis from computed tomography scans.

Bone maturation is the only reliable indicator of growth and its radiologic assessment with or without automated systems is a qualitative method. Image processing allows the study of bone maturation with quantitative data. Carpal bone maturation was studied in 20 children (13 boys and 7 girls, ages ranging from 4 to 15 years) without any clinical evidence of endocrine disease by image analysis from computed tomography (CT) scans. Each wrist CT scan was processed in order to extract the carpal bones and to measure quantitative data regarding volume, axes of inertia and density for each bone. The volumes and the length of the inertia axes were significantly correlated with age. Whatever the age, there were strong correlations between the volume or the length of the main inertia axis of one carpal bone and that of all others. The decrease in the carpal bone volume measured from the processing procedure compared with the theoretical volume of bone defined from the length of the three inertia axes indicated a change in bone shape during growth. Although the mean density was constant, there was an increase in the standard deviation of density with age. Skeletal maturity assessment with image analysis from CT scans seems to be a good complementary investigation to determine bone age in children.

Adolescent↗

Image analysis and computer vision in medicine.

Multimedia lives with images; medical images are born from digital imaging. A physician's multimedia workstation cannot exist without tools for manipulating images, performing measurements and, generally speaking, extracting and collecting pieces of information from the available data. Image analysis and computer vision constitute a wide and rapidly evolving field. This paper is intended as an introductory document for medical imaging researchers and practitioners wishing an overview of recent developments and trends. The major lines of activities in the domain are presented, under the common framework of a processing pipeline. After a presentation of the various stages of this pipeline, current subjects of research are indicated.

Computer Graphics↗

Rapid and simple quantitative measurement of alpha-fetoprotein by combining immunochromatographic strip test and artificial neural network image analysis system.

BACKGROUND: Quantitative immunochromatographic strip (ICS) assay can facilitate clinical diagnosis by providing more information than traditional qualitative or semiquantitative strip assay. METHODS: We constructed a human serum alpha-fetoprotein (AFP) measurement system by combining semiquantitative ICS tests and artificial neural network (ANN) image analysis system [immunochromatographic strip analyzed by artificial neural network image analysis system (IAIS)]. After ICS tests completed, the AFP concentration can be obtained from analysis of IAIS software. The equipment required are commercial semiquantitative AFP dipstick, optical scanner, centrifuge (option), personal computer, and software of IAIS. RESULTS: The serum AFP concentrations measured by IAIS were strongly correlated (r = 0.9971) with that by RIA. Using Bland-Altman analysis, the IAIS achieved clinical acceptable limits of agreement in comparison with RIA. The within-run precision of IAIS, expressed as coefficients of variation (CV), at 81.7 ng/ml was 1.50% and, at 244.4 ng/ml, was 1.09%. The measurement of serum AFP by IAIS can be completed in 20 min. CONCLUSIONS: The newly constructed quantitative immunochromatographic strip assay (IAIS) is a simple, rapid, and reliable method for serum AFP measurement. With the simple equipment required, the IAIS can be performed outside the laboratory and is ideal for outpatient or point-of-care AFP testing.

Biomarkers, Tumor↗

Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells.

Feulgen-DNA content, nuclear phenotypes, and levels of chromatin condensation were evaluated by image analysis in NIH/3T3 cells transformed with the c-H-ras oncogene of T24 cells. Three nuclear phenotypes, differing from those of untransformed control cells and defined in terms of patterns of chromatin condensation, were demonstrated microspectrophotometrically for the tumor cells. Polyploidy could only be observed in nuclei with extensive and deeply stained areas covered with condensed chromatin, i.e., only in a small fraction of the tumor cell nuclear population. The increased chromatin condensation that appeared with cell transformation affected the euchromatin zones. The image analysis provided data that, compared with those obtained in other situations involving cell transformation, could be relevant to the understanding of changes in chromatin supraorganization related to tumorigenesis and to tumor cell diagnosis.

Animals↗

Endpoint markers for cancer chemoprevention trials derived from the lesion of precancer (intraepithelial neoplasia) measured by computer-assisted quantitative image analysis.

Endpoint markers for cancer chemoprevention clinical trials are described that are developed from the morphological properties of the precancerous lesion of intraepithelial neoplasia itself, as measured by computer-assisted quantitative image analysis. The markers include increased proliferative fraction (percentage MIB-1 positive nuclear area); nuclear DNA content (DNA ploidy), including DNA content exceeding fivefold the haploid amount (5C-exceeding rate); nuclear/nucleolar morphometry; and disorganization of nuclear chromatin pattern as characterized by Markovian parameters and other functions. A significant new advance in image analysis is the process of "tiling," in which hundreds of full monitor image fields of a given histological section at x40 magnification are reduced in size and fused seamlessly to produce a single image of the histological section at x1.25 magnification. The operator may review the low-power image and retrieve x40 magnification of any desired area by point/clicking with a mouse.

Algorithms↗

Image analysis of the histochemical demonstration of glucose-6-phosphatase activity in rat liver.

Activities of histochemically demonstrated glucose-6-phosphatase were quantified by computerized densitometry using image analysis in livers of female adult Wistar rats fed ad libitum and fasted 22 h before sacrifice. Mean optical densities along the path between small portal tracts and efferent hepatic venous branches and enzyme activities obtained from biochemical assays exhibited a strong positive correlation. The gradients of high periportal to lower perivenous glucose-6-phosphatase activities were analysed by profiles of optical density along these distances. Mapping optical densities in an image of equidensity range provided information on the distribution pattern of hepatic glucose-6-phosphatase over an extended two-dimensional area. This visualisation of histochemical enzyme reaction based on quantitative data supports the approach of sampling across the entire protal----hepatic venous distance disregarding parenchymal zonation. Utilities provided by computer assisted image analysis will have some bearing for further adequate quantitative description of liver function and structural make up.

Animals↗

Histomorphometric analysis: comparative study of manual and digital image analysis using reproductive organ as a model.

A preliminary analysis of the histological features of testes was carried out on vertical serial sections stained with hematoxylin and eosin techniques. The total spermatogonia per view and areas of seminiferous tubules by means of digitizing the tissue followed by morphometric analysis using software run on a personal computer. The data generated was then compared with traditional manual determinations made by using the ocular scale as well as the x and y coordinated on the light microscope. The data obtained showed that the modified Image Pro system for digitizing and data analysis allowed for high-resolution acquisition of the images as well as relatively high sensitivity of image integration. The results also show more accuracy and repeatability of sampling and a decrease in sampling error between repeated measures compared with the conventional manual measurements. Overall, the use of modified Image Pro digital technology will offer a more accurate and quantitative method to perform routine histomorphometric analysis, as well as allow for repeated measurements of the same sample from several reviewers in a blinded fashion with substantially less error.

Animals↗

Computer image analysis of the optic disc.

A computer method for image analysis of the optic disc is presented. Black and white photographic transparencies of the optic disc were scanned by a scanning television camera. The image was digitized into not more than 4,000 x 4,000 points, each representing a value ranging between 0 and 255 gray levels. The images are depicted in two ways; either in the form of a topographic map (contour) in which the curves join points of identical light intensity, or as a relief that expresses the light intensity of every scanned line. This method emphasizes the qualitative parameters of the disc, and enables a quantitative determination of some disc pathologies.

Computers↗

Changes in intestinal tunica muscularis following dietary fiber feeding in rats. A morphometric study using image analysis.

The morphological changes in the intestinal tunica muscularis induced by prolonged dietary fiber intake were determined in rat small intestine and colon with the aid of computerized image analysis. Thirty male Sprague-Dawley rats were fed either a fiber free, 15% cellulose or 15% pectin diet for 8 weeks. Intestine length was measured and stained cross sections of the jejunum, ileum, and colon were quantitated using image analysis. In the distal colon, muscle cell size was also determined. Despite lower weight gain in the pectin fed rats, both the small intestine and colon length were significantly increased. Cellulose feeding had a lesser effect on intestine length. Pectin fed rats had significantly increased relative tunica muscularis area (37.2 +/- 2.2 mm2) in ileum cross sections when compared to control (24.3 +/- 1.8 mm2) and cellulose fed rats (26.1 +/- 1.1 mm2). In the mid-colon, the tunica muscularis area was found to be pectin > cellulose > control (33.5 +/- 2.2; 29.7 +/- 1.7; 25.8 +/- 1.5 respectively) with significant differences reached between pectin and control rats. In jejunal samples, no differences were observed among the groups. Circular smooth muscle cell size in the distal colon was significantly increased following cellulose feeding but was less pronounced in the case of pectin. We conclude that fiber supplementation leads to morphological changes in the rat intestine including changes in length and tunica muscularis volume.

Animals↗

Study of the connectivity properties of Bioglass -filled polylactide foam scaffolds by image analysis and impedance spectroscopy.

The porous structure of two series of poly(D,L-lactide)/Bioglass composite foams prepared by thermal-induced phase separation was investigated by image analysis and impedance spectroscopy. Polymer solutions of either low or high molecular weight containing different concentrations (up to 50 wt.%) of Bioglass particles of mean particle size d < 5 microm were studied. The morphology of both macro- and micropores was studied by scanning electron microscopy and image analysis of both neat and composite foams (containing 10-50 wt.% Bioglass). The pore connectivity of both neat polymer and composite foams was characterized by impedance spectroscopy in relation with their transport properties. The influence of the foam composition (i.e., polymer molecular weight and concentration of Bioglass on pore microstructure was studied using these non-destructive methods. It was found that addition of Bioglass particles has a pronounced effect on pore orientation, leading to increasing loss of order of pore structure, especially for low-molecular weight PDLLA foams.

Biocompatible Materials↗

Comparison of morphological characteristics of Streptomyces natalensis by image analysis and focused beam reflectance measurement.

A morphological interpretation is presented for data collected during growth of a filamentous organism, using a focused beam reflectance measurement (FBRM) system. The morphology of the organism was also obtained using conventional semiautomatic image analysis to support the interpretation of the FBRM data. The model organism employed is the filamentous soil-borne actinomycete Streptomyces natalensis, which produces the antifungal agent pimaricin. The organism was cultivated both in shake flasks and in a bench-scale stirred tank bioreactor. It was found that FBRM could be used to track changes in the morphology of the organism throughout the course of its growth on both scales. These changes were highlighted using both the median chord length and length-weighted mean chord length obtained from the chord length distribution measured with the FBRM probe. The ability of the FBRM probe to respond to changes in both the size and morphology of mycelial aggregates was supported by standard image analysis parameters, including equivalent diameter, convex area, and compactness.

Bioreactors↗