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Characterization of bacterial growth on solid medium with image analysis.

Alkaliphilic bacterial strains producing the enzyme cyclodextrin glucanotransferase were cultivated on solid agar medium containing an indicator system detecting the enzyme. The growth of the colony and the surrounding diffusion zone, due to the enzyme, were measured by the image analysis during the cultivation. It was possible to differentiate between relatively similar clones by observing quantitatively the changes at and around the colony. Optimal experimental conditions for such measurements are discussed. The image analysis technique provides a potential tool for characterizing microbes grown on solid media.

Bacteria↗

Quantifying the smooth muscle content of the prostate using double-immunoenzymatic staining and color assisted image analysis.

The primary objective of the present study was to develop a method for quantifying the smooth muscle content of the prostate adenoma. A double immunoenzymatic staining technique was coupled with color assisted image analysis to determine the area density of the smooth muscle within the prostate adenoma. Eight males with symptomatic BPH underwent transrectal biopsy of the prostate. Four micron thick tissue sections were used for the double immunoenzymatic staining process. Rabbit anti-desmin and mouse anti-human prostatic acid phosphatase antibodies were used to selectively bind smooth muscle and prostatic epithelium, respectively. The two different tissue antigens were identified with peroxidase-antiperoxidase (PAP) and alkaline phosphatase-antialkaline phosphatase techniques. The alkaline phosphatase activity and peroxidase activity were developed with fast red and DAB chromogens. The BQ MEG IV Vista color system image analysis was used to discriminate color differences from the stained tissue sections. The thresholds were set to identify smooth muscle (dark brown), epithelium (red), fibrous tissue (pale brown), and glandular lumina (colorless). The mean area density of smooth muscle, fibrous tissue, glandular epithelium, and glandular lumina was 22%, 54%, 16%, and 9%, respectively. The present study suggests that a significant component of the prostate adenoma is smooth muscle. The application of this technique will be utilized to provide further insights into the role of smooth muscle in the pathogenesis and therapy of BPH.

Aged↗

Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system.

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.

3,3'-Diaminobenzidine↗

Desktop computer-based image analysis of cell surface fluorescence patterning from a photographic source.

We report the use of standard computer-based image analysis technology to analyze, from a photographic source, individual cell surface receptor site patterns using fluorochrome labeling. The model used in this study was a Strongylocentrotus purpuratus sea urchin embryo labeled with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA) (0.5 mg/ml for 5 min at 15 degrees C incubated with fertilization membrane free embryos). Image capture was performed using desktop-type digital scanning, and the images were imported into Adobe Photoshop for study. All images analyzed were derived from a single photographic negative: 1) the actual micrograph printed from the negative and scanned into a Macintosh IIx computer; 2) the scanned negative itself; and, 3) a high resolution scanning process used with a Kodak Photo CD. Patterns were analyzed using the densitometry feature of Photoshop, and were similar enough from all 3 scanned images to indicate that any of the 3 scanning processes can be used for fluorescence patterning analysis. Adobe Photoshop was also used to highlight, for closer analysis, the fluorescence patterns by producing 3-D effects, border mapping and transition area detailing. The desktop image analysis procedures described here to study fluorescence patterning require no expensive scientific hardware or software.

Animals↗

An image analysis on pathological changes in pulmonary arteries in chronic obstructive pulmonary disease.

The changes in small pulmonary arteries of 15 patients with chronic obstructive pulmonary disease (COPD) were investigated by light and electron microscopy, image analysis etc. It was found that the structural changes in the pulmonary arteries of the patients with COPD were characterized by muscularization of non-muscular arterioles, media hypertrophy, longitudinal smooth muscle bundles in the intima and fibrosis in both the media and intima. In the course of time, these lesions resulted in thickening of the arterial wall and narrowing of the lumen. Clinically, the patients developed pulmonary hypertension causing cor pulmonale. Initial data on the structure of arterial wall at different segments were compared statistically. There was very significant difference between the COPD and control groups (P less than 0.001). By Fisher's auto-classification (automatic pattern recognition), the rate recognized was correct in more than 90% in the small arteries of less than 200 microns in diameter. It is suggested that these arteriolar changes are closely related to pulmonary hypertension. The image analysis showed that the ratios of MWA/MVA and MWT/MD were of great value in evaluating the degree of the changes in the arteries of the patients with COPD.

Female↗

Examination of the glycocalyx of four species of Staphylococcus by transmission electron microscopy and image analysis.

The coagulase-negative staphylococci have become important pathogens in human infections involving foreign bodies. The bacterial glycocalyx is a major mediator of attachment of these organisms to medical devices, but the glycocalyx is sometimes difficult to demonstrate. A combination of the techniques of transmission electron microscopy (TEM) and image analysis enabled investigators to reveal the glycocalyx which was previously indiscernible. Eight strains of coagulase-negative staphylococci, including Staphylococcus epidermidis, S. hominis, S. lugdunensis, and S. schleiferi subspecies schleiferi, were grown, treated with anti-staphylococcal serum to stabilize the glycocalyx, and examined by TEM. Image analysis of negatives was then used to enhance the visual images which showed far more glycocalyx than previously seen by TEM alone.

Bacterial Capsules↗

[Pattern recognition of stereoscopic features of the leaves epidermis of medicinal Curcuma plants in China by image analysis].

OBJECTIVE: To provide new ways for classifying and identifying medicinal curcuma plants. METHOD: Based on classical taxonomy, the microscopic features of the epidermis of Curcuma plants in China were scored quantitatively by stereology and image analysis. RESULT: It showed that there were a lot of differences in density, size and shape of the epidermic cells among the medicinal Curcuma plants in China; that the average perimeter of the upper epidermic cells, average sectional area of the lower epidermis, stoma density and trichoma distribution of the upper and lower epidermis, etc. could be considered to be the main evidences for the microscopic identification of leaves of Curcuma. The 21 materials which belong to 11 species of Curcuma in China were systematized into 6 species, 1 species complex, 2 cultivated varieties. CONCLUSION: It can be concluded that stereology, and image analysis are advanced and feasible in pharmacognosy and taxonomy especially in the authentication of the relative and easily confused species.

Curcuma↗

Measurement of epidermal thickness in a patient with psoriasis by computer-supported image analysis.

The aim of the present study was to measure full epidermal thickness, stratum corneum thickness, rete length, dermal papilla widening and suprapapillary epidermal thickness in psoriasis patients using a light microscope and computer-supported image analysis. The data obtained were analyzed in terms of patient age, type of psoriasis, total body surface area involvement, scalp and nail involvement, duration of psoriasis, and family history of the disease. The study was conducted on 64 patients and 57 controls whose skin biopsies were examined by light microscopy. The acquired microscopic images were transferred to a computer and measurements were made using image analysis. The skin biopsies, taken from different body areas, were examined for different parameters such as epidermal, corneal and suprapapillary epidermal thickness. The most prominent increase in thickness was detected in the palmar region. Corneal thickness was more pronounced in patients with scalp involvement than in patients without scalp involvement (t=-2.651, P=0.008). The most prominent increase in rete length was observed in the knees (median: 491 microm, t=10.117, P=0.000). The difference in rete length between patients with a positive and a negative family history was significant (t=-3.334, P=0.03), being 27% greater in psoriasis patients without a family history. The differences in dermal papilla distances among patients were very small. We conclude that microscope-supported thickness measurements provide objective results.

Adolescent↗

Selection of embryogenic sugarcane callus by image analysis.

In the cultivation of plant calli on solid media, two kinds of calli such as compact and friable calli, which are a bright yellow and a whitish clump, respectively, are often obtained. Distinction of these calli is of much importance in the regeneration step. The image analysis system associated with a Charge Coupled Device (CCD) camera and microscopy were used to distinguish sugarcane calli. The original images from compact and friable calli were input to a computer via an image analysis board. At first, the brightnesses of trichromatic colors, red (R), green (G) and blue (B), of each pixels were extracted and the average brightness value for each color was calculated. From these values of the trichromatic colors, compact and friable calli could not be clearly distinguished. Next, the brightness of yellow, Br(Y), and white, Br(W), were defined using Br(R), Br(G) and Br(B), and the difference between Br(Y) and Br(W), Br(Y-W), which can be used to express the yellowish grade, was calculated. When Br(Y-W) was determined from all pixels of the original images of both calli, the compact calli were found to be clearly distinguished from the friable calli by the frequency distributions of Br(Y-W). Average brightness center value, Av(C(Y-W)), was calculated from the frequency distributions. It was found that the calli with less than 10 units of Av(C(Y-W)) was never regenerated and a proportional relationship between Av(C(Y-W)) and the regeneration frequency of the callus line was obtained.

Journal Article↗

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

BACKGROUND: To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. METHODS: We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. RESULTS: Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. CONCLUSIONS: The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.

Cell Division↗

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Animals↗

Anti-CagA antibodies are associated with atrophic gastritis in a population at high gastric cancer risk: a morphometric study by computerized image analysis.

BACKGROUND: CagA-positive Helicobacter pylori strains appear to increase the risk for atrophic gastritis. AIM: To verify the association between CagA status and atrophic gastritis in the general population by means of computerized image analysis. SUBJECTS: Forty-five subjects were chosen out of a representative sample of a population at high gastric cancer risk. METHODS: Helicobacter pylori status was assessed by IgG ELISA, rapid urease test and histology. Serum anti-CagA antibodies were detected by western blotting. Subjects were subdivided into 3 groups: 15 subjects Helicobacter pylori positive CagA-positive, 15 Helicobacter pylori positive CagA-negative and 15 controls Helicobacter pylori negative. Biopsies were studied using the Sydney System score. A computerized image analysis was used to count inflammatory cells in the lamina propria and to measure the area of the gastric glands. RESULTS: Anti-CagA antibodies were associated with reduction of gland area (126,671 +/- 81,032 mu 2/mm2 vs 231,384 +/- 54,159; p = 0.0001), with increasing both of polymorphonuclear cells (426 +/- 238 cell/mm2 vs 136 +/- 69; p = 0.0001) and mononuclear cells (8675 +/- 1304 cell/mm2 vs 7141 +/- 1230; p = 0.003). CONCLUSIONS: The association of anti-CagA antibodies with a high grade of gastric atrophy further supports the hypothesis that Helicobacter pylori CagA-positive strains can promote the multifactorial process of gastric carcinogenesis.

Adult↗

Determination of adipocyte size by computer image analysis.

Methods that allow rapid and accurate determination of adipocyte size are important to studies of energy and glucose metabolism. The direct measurement of adipocyte size by microscopy is widely used, although the method is tedious and time consuming. Computer-assisted image analysis can overcome most of the disadvantages associated with this technique. We report a new method for determining adipocyte size by measuring the cross-sectional area of adipocytes with computer image analysis. This method allows a large number of adipocytes to be measured rapidly with computer hardware and software that are readily available.

Adipocytes↗

DNA content by image analysis. An accurate discriminator of malignancy in pericardial effusions.

DNA analysis was performed on 20 cytologically equivocal pericardial effusions to determine if it would be a useful ancillary technique in identifying malignancy. Ploidy determination by interactive image analysis showed single diploid DNA peaks, with no cells > 5c, in all eight reactive cases studied. The reactive cases had a mean proliferation index of 1.3. All 12 malignant cases studied had cells with DNA content > 5c. A dominant DNA peak at 2c was present in 25%, dominant aneuploid stem lines were present in 25%, and multiple stem lines were present in 50% of the malignant cases. By image analysis, the presence of DNA content > 5c was both sensitive and specific for detecting malignancy in the group of pericardial effusions evaluated.

Cell Count↗

Application of automatic image analysis (IBAS system) to age calculation. Efficiency in the analysis of several teeth from a single subject.

In a previous paper (M. López-Nicolás et al., Forensic Sci. Int., 45 (1990) 143-150) we obtained a model of multiple regression to estimate age using computerized image analysis (IBAS) to study teeth. This method should provide more precise measurements of the parameters involved and to test this hypothesis we have carried out a blind study using more than one tooth from the same individual in order to check the diagnostic accuracy of the method. Our results show that individual variability introduced serious errors in tooth age estimation using computerized image analysis. However the study of more than one tooth per individual improves the accuracy of age estimation. Specific cases should therefore be interpreted cautiously, as a degree of variability can be expected using the parameters defined in this method.

Adult↗

Preliminary study of hair form of Japanese head hairs using image analysis.

The use of average curvature measurements for the forensic comparison of curly hairs has been reported, but a method, in which various types of hair form are quantitatively examined and objectively interpreted for hair comparison, has not been reported to date. In the present study, numerical data on hair form from Japanese subjects were obtained by image analysis and a morphological comparison of these head hairs was investigated. Head hairs obtained from eight Japanese males were measured for length (L), distance (D) and area (A) using a Kontron Imaging System KS400. From the three measurements mentioned above, three indexes, L/D, A/D and 2(A/L), were examined. The inter-individual variations for each value were investigated by a t-test and the availability of six values for the forensic comparison of hair form was evaluated by a stepwise linear discrimination analysis. Six values obtained from hair form by an image analysis showed large intra-individual variations. However, these six values were found to be useful for discriminating between two individuals, since the six values showed larger inter-individual variations than intra-individual variations. Discrimination on each comparison using a stepwise linear discrimination analysis was performed for some of the values and the results indicated conspicuous inter-individual variations between the two individuals. On 11 of 28 comparisons, 30 hairs from one individual could be completely distinguished from hairs of another individual, when a two-way comparison was employed. These results confirm that hair form could be quite useful in the forensic comparison of hair morphology, and suggest that numerical data obtained from hair form by image analysis are very important values for constructing a screening procedure for evidential hairs. The use of an objective measure of hair form will be especially useful for Japanese head hairs since they are generally thought to show very limited variation in morphological features.

Asian People↗

[Chemotherapy for metastatic brain tumors with CDDP and other agents: correlation between chemotherapeutic effects and the results of in vitro chemosensitivity tests using collagen gel-embedded culture combined with computerized image analysis in metastatic brain tumors].

Chemotherapy with CDDP and/or other agents was performed in 15 patients after removal of metastatic brain tumors. A chemosensitivity test using a system of collagen gel-embedded culture and computerized image analysis was performed on the tumors from these patients. The clinical usefulness of the chemosensitivity test was evaluated by comparing chemotherapeutic effects with the results of the test. The rates of correlation of the chemosensitivity test with clinical response on brain MRI was 80%, and that of the chemosensitivity test with clinical response in tumor markers or on primary tumors was 75%. This observation suggests that the chemosensitivity test using collagen gel-embedded culture and computerized image analysis is useful in determining optimal chemotherapy for metastatic brain tumors. In ten multiple metastatic brain tumors, three complete responses, two partial responses, one minor response and four non-responses were observed on MRI. Only one case showed a false negative result on the chemosensitivity test and showed partial response. This result also indicates the effectiveness of chemotherapy based on chemosensitivity testing.

Adenocarcinoma↗

Image analysis of gunshot residue on entry wounds. II--A statistical estimation of firing range.

A statistical investigation of the relationship between firing range and the amount and distribution of gunshot residue (GSR), used automated image analysis (IA) to quantify GSR deposit resulting from firings into pig skin, from distances ranging between contact and 45 cm. Overall, for a Ruger .22 semi-automatic rifle using CCI solid point, high velocity ammunition, the total area of GSR deposit on the skin sections decreased in a non-linear fashion with firing range. More specifically there were significant differences in the amount of GSR deposited from shots fired at contact compared with shots fired from distances between 2.5 and 45 cm; and between shots fired from a distance of 20 cm or less, with shots fired at a distance of 30 cm or more. In addition, GSR particles were heavily concentrated in the wound tract only for contact and close range shots at 2.5 cm, while the particle distribution was more uniform between the wound tract and the skin surfaces for shots fired from distances greater than 2.5 cm. Consequently, for future scientific investigations of gunshot fatalities, once standards have been established for the weapon and ammunition type in question, image analysis quantification of GSR deposited in and around the gunshot wound may be capable of providing a reliable, statistical basis for estimating firing range.

Animals↗