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PubMedDate not supplied

Image analysis techniques for characterizing disc space narrowing in cervical vertebrae interfaces.

Image analysis techniques are introduced for evaluating disc space narrowing of cervical vertebrae interfaces from X-ray images. Four scale-invariant, distance transform-based features are presented for characterizing the spacing between adjacent vertebrae. K-means and self-organizing map clustering techniques are applied to estimate the degree of disc space narrowing using a four grade (0-3) scoring system, where 0 and 3 represent normal spacing and significant narrowing, respectively. For a data set of 294 vertebrae interfaces, experimental results yield average correct grade assignment of greater than 82.10% for each of the four grades using a one grade window around the correct grade.

Algorithms↗
PubMed1987

CYBEST Model 4. Automated cytologic screening system for uterine cancer utilizing image analysis processing.

CYBEST (Cyto-Biologic Electronic Screening System) utilizes image analysis technology for the automated prescreening of cervical cytology specimens. CYBEST Model 3, which includes a television scan system and automatic shading control, achieved our initial goal of rapid specimen processing (no more than three minutes to achieve a final specimen assessment). This paper describes CYBEST Model 4, developed in 1981; with the minicomputer of Model 3 replaced by a microcomputer, Model 4 is considerably smaller, about the size of a business desk. A new parameter, the intranuclear configuration (chromatin pattern), was added to the four parameters used in Model 3. The five parameters now used for the assessment and ranking of cytologic abnormalities are nuclear size, nuclear-cytoplasmic ratio, nuclear optical absorption, nuclear shape and intranuclear configuration. The other features of Model 4 are almost the same as those of Model 3. As an optional function, individual parameter measurement data, assessment of atypicality grade and cell images can be displayed on the CRT monitor by pointing to a cell with a light pen system. After completion of screening of a specimen, the ten cells judged to be most abnormal can be called automatically into the microscope optical field or the CRT monitor (in order ranging from the cell with the highest atypicality rank down) along with their associated data and the system's assessment of the specimen. By connection to a small business computer, all data can be transferred to a floppy disk for later retrieval.

Cytodiagnosis↗
PubMedDate not supplied

Measurement of DNA content and nuclear pleomorphism in metastatic variants of the B16 murine melanoma and hamster lymphoma and its liver metastasis using image analysis techniques.

The DNA content and nuclear pleomorphism (NPM), which are two cellular features consistently employed in the assessment of tumour malignancy, have been measured in B16 murine melanoma metastatic variants and in hamster primary lymphoma and its liver metastasis as tumour models using image analysis techniques. The three melanoma variants studied were the low metastasis variant F1, the BL6 variant selected for high lung metastasis and invasive ability, and ML8, a line isolated from pulmonary metastasis of the BL6 tumour. The cellularity of the melanomas bore no relationship to metastatic ability. The cell cycle distribution of nuclei based on integrated nuclear density (IND) was studied. The ML8 tumour showed higher DNA ploidy. Also, in this tumour the S-phase fraction was approximately 2.0-fold larger than that of the BL6 tumour. Flow cytometry of nuclei isolated from paraffin-embedded tumour tissue showed all three melanomas were aneuploid. In both F1 and BL6, two distinct subpopulations (p2 and p3) of nuclei, based on the degree of their pleomorphism, could be discerned. A significantly higher proportion of the more pleomorphic subpopulation (p2) occurred in BL6 than in F1. In the ML8 alone, a third subpopulation (p1), which was more pleomorphic than p2, was found. The hamster lymphomas (HALY-malignant and N-HALY-non-malignant) were less cellular than the metastatic tumour (HALY-met) in liver. The lymphomas N-HALY and HALY-met had a higher DNA ploidy as compared with its primary tumour HALY. However, the non-malignant lymphoma N-HALY and the moderately malignant hamster fibrosarcoma were also found to be hyperdiploid. The metastatic lymphoma (HALY-met) showed a more pleomorphic nuclear subpopulation as compared with the primary. No differences were found in the size of the S-phase fractions of the hamster tumours. The present work shows that image analysis techniques enable one to make objective measurements of DNA content and nuclear pleomorphism of tumour cells, and suggests that in the tumour models investigated there is increased nuclear pleomorphism and DNA ploidy associated with tumour progression.

Animals↗
PubMed1991

Image analysis of microsialograms of the mouse parotid gland using digital image processing.

We compared two digital-image feature-extraction methods for the analysis of microsialograms of the mouse parotid gland following either overfilling, experimentally induced acute sialoadenitis or irradiation. Microsialograms were digitized using a drum-scanning microdensitometer. The grey levels were then partitioned into four bands representing soft tissue, peripheral minor, middle-sized and major ducts, and run-length and histogram analysis of the digital images performed. Serial analysis of microsialograms during progressive filling showed that both methods depicted the structural characteristics of the ducts at each grey level. However, in the experimental groups, run-length analysis showed slight changes in the peripheral duct system more clearly. This method was therefore considered more effective than histogram analysis.

Animals↗
PubMed1992

Autocrine and paracrine effects of endothelium-derived relaxing factor on intracellular Ca2+ of endothelial cells and vascular smooth muscle cells. Identification by two-dimensional image analysis in coculture.

To elucidate the effects of endothelium-derived relaxing factor (EDRF) released from vascular endothelial cells (ECs) on handling of intracellular calcium ion (Ca2+i) in ECs themselves and vascular smooth muscle cells (VSMCs), we measured the Ca2+i by two-dimensional digital image analysis of fura-2-loaded ECs and VSMCs in tissue culture. In isoculture of one cell type, adenosine triphosphate (ATP, 1 microM) transiently increased the Ca2+i of both ECs and VSMCs. High-K+ depolarization or angiotensin II also elevated the Ca2+i of VSMCs, whereas neither stimulants changed the Ca2+i of ECs. In coculture of ECs with VSMCs, the same dose of ATP rapidly increased the Ca2+i of ECs and then transiently decreased the Ca2+i of VSMCs to below the resting level. The maximal Ca2+i-modulating effects of ATP on both cell types were reproducible after the second application of ATP. Three kinds of EDRF blockers (L-NG-monomethylarginine, methemoglobin, or methylene blue) potentiated the ATP-induced Ca2+i rise in ECs and attenuated the Ca2+i reduction in VSMCs, suggesting the autocrine and paracrine effects of EDRF on ECs and VSMCs, respectively. However, neither indomethacin, superoxide dismutase, nor neutralizing monoclonal antibody to endothelin-1 altered the second responses. Thus, two-dimensional Ca2+i image analysis of ECs and VSMCs in coculture enabled direct visualization of the EDRF actions in ECs and VSMCs and their modifications.

Adenosine Triphosphate↗
PubMed2000

Quantitative image analysis of lung connective tissue in murine silicosis.

Pulmonary fibrosis is a disabling consequence of many lung diseases but is difficult to quantify. Lucifer yellow CH fluorescent dye (LY) appears to stain connective tissue matrix macromolecules selectively. Laser scanning confocal microscopy can quantify the intensity of fluorescence and determine the area of fluorescent material. We hypothesized that the abundance of lucifer yellow-stained matrix macromolecules in lung tissue sections could be measured by laser scanning confocal microscopy, would reflect differences between varying degrees of pulmonary fibrosis, and could be compared directly to biochemical measurements of lung collagen. We exposed C57B1/6 and 129 strains of mice by aerosol to cristobalite silica (70 mg/m3, 12 days, 5 hours/day) or sham-air and examined them 2 and 16 weeks after exposure. The area of LY-stained matrix in tissue sections was quantitated by laser scanning confocal microscopy, and total lung collagen was measured biochemically as hydroxyproline (OH-proline). The LY-stained connective tissue matrix appeared as bright linear bands in the alveolar septae, and was increased significantly by image analysis in C57B1/6 and 129 mice with silicosis 16 weeks after exposure. Total lung OH-proline was significantly increased in silica-exposed mice from both stains at both time points. Comparing all 8 groups, there was a significant linear correlation between the average area of connective tissue measured by LY stain and the total OH-proline per lung measured by chemical analysis (r = .72, P = .042). LY staining and confocal microscopy with image analysis offers a rapid technique for quantitative measurements of the extent of pulmonary fibrosis.

Animals↗
PubMed2002

Quantification of erythema using digital camera and computer-based colour image analysis: a multicentre study.

BACKGROUND/PURPOSE: Colour measurements obtained from digitized images have been proposed as a simple and cost-effective way to evaluate skin colour and the activity of treatments. The main disadvantage of the method is the fact that it is highly dependent on ambient light: even if an accurate control of subjects' illumination is provided, readings remain not comparable among different laboratories. The purpose of this study was to develop a highly reproducible system for computerized colour image analysis of skin erythema, making it possible to compare readings from different environmental light conditions. PATIENTS AND METHODS: Three hundred and forty-eight Caucasian adult healthy subjects (age range: 18-60 years) of both sexes (14% males, 86% females), were enrolled in the study by 49 dermatologists distributed all over Italy. They were recruited among patients who required aesthetic treatments involving skin erythema, like chemical peeling and laser epilation. Once the treatment was administered, clinical evaluations and pictures were taken at the level of treated areas. Visual assessment of erythema was done on the basis of conventional clinical grades (0 = absent; 1 = slight; 2 = moderate; 3 = intense). The clinicians participating in the study were asked to put a standard colour marker (red, green and blue coloured self-adhesive ring) in the photographed skin area. The difference between r, g, b values of photographed colour markers on the skin of single patients participating in the study and the r, g, b values obtained photographing the colour marker in fixed illumination conditions was used to adjust skin colour measurements. Then erythema index (E.I) on digitized images was calculated subtracting red value to green one by averaging procedure of different pixels. RESULTS: Erythema index. average value among the groups divided according to the conventional clinical score increased progressively from score 0-2, while it decreased from score 2 to score 3. The differences in E.I. mean values among the score groups (0 vs. 1, 1 vs. 2, 2 vs. 3) were statistically significant (P < 0.05). CONCLUSION: We developed a method for the measurement of skin erythema using digital camera, normalized r, g, b colour co-ordinate system and computerized calculation of E.I. Clinical usefulness of our method for absent, slight and moderate erythema, was demonstrated. For intense erythema lesions we did not find a correspondence between clinical and computerized evaluation, probably due to other factors involved in skin inflammation (e.g. oedema).

Adolescent↗
PubMed1992

Noncolorimetric measurement of cell activity in three-dimensional histoculture using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide: the pixel image analysis of formazan crystals.

We describe a novel system for measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction in three-dimensional histoculture which is no longer dependent on colorimetric determination of extracted formazan, but rather is based on a pixel image analysis of formazan crystals, and which allows intratumor heterogeneity to be taken into account. The MTT test is based on the enzymatic reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide to formazan crystals by living, metabolically active cells, but not in dead cells. The reaction was carried out in situ in six-well plates on gel-supported histocultured human tumors. After a 24-h incubation with different drugs the tumors were incubated with a solution of MTT. Frozen sections of the tumor pieces were made and the slides were then stained with a propidium iodide solution, whose fluorescence is proportional to the number of cells present. We demonstrate here that the formazan crystals, formed by MTT reduction, reflect polarized light and that this can be quantified by using an image analysis system based on bright-pixel quantitation directly on a frozen section of the original tissue. Combined with the use of the fluorescent dye propidium iodide, also measured by pixel analysis, we can express a ratio between the total amount of MTT reduction and the total number of cells present in the specimen that expresses the effect of drugs on the histocultured tumors. Since histology is well maintained in histoculture it is possible to take into account the heterogeneity present in the tumor with regard to drug response.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1994

Ploidy of 36 stromal tumors of the gastrointestinal tract. A comparative study with flow cytometry and image analysis.

DNA ploidy was investigated by flow cytometry (FC) and image analysis (IA) in paraffin-embedded tissue sections from 36 stromal tumors of the gastrointestinal tract. The results of both techniques were correlated with pathologic features of the tumors and survival. Ten (27.8%) tumors were aneuploid by FC and IA. Most of the diploid tumors were identified by both techniques, but FC appeared to be superior to tissue section IA for identification of aneuploid tumors (25% vs. 13.8%). Aneuploidy by FC correlated with pathologic grade and mitotic index (P < .05), and a trend to short survival was also detected (P < .1). No similar correlation was found by IA. Enlargement and variation of nuclei may explain the discrepancy between FC and IA.

Flow Cytometry↗
PubMed2002

Diagnostic approach of effusion cytology using computerized image analysis.

The objective of this study is to investigate whether image cytometry is a sensitive and specific method for the differential diagnosis of equivocal cells in routine cytology of effusion smears. One hundred four effusion smears were studied from routine cytologic material. Cytologically 56 (53.8%) of the smears were classified as malignant, 26 (24%) as suspicious and 22 (21.1%) as benign. Two morphometric variables (nuclear major axis length and nuclear area) of the nuclei were measured by an image analysis system. Higher values for the area were found for malignant rather than benign and suspicious cells (p < 0.0005 and p < 0.005 respectively). The same result was extracted for the nuclear major axis length values (p < 0.0005 and p < 0.0005 respectively). Values of nuclear major axis length and nuclear area didn't differ significantly between benign and suspicious cells (p = 0.071 and p = 0.066 respectively). The results show that the range of the values for suspicious cells is closer to the range of the benign cells. Cytomorphometry of the effusion smear cells may provide important information for the differentiation of atypical mesothelial cells from malignant adenocarcinoma cells.

Adenocarcinoma↗
PubMed1997

Groping for quantitative digital 3-D image analysis: an approach to quantitative fluorescence in situ hybridization in thick tissue sections of prostate carcinoma.

In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH). The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA) can be estimated by counting the number of FISH signals per cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI) to a software package for display, inspection, count and (semi-)automatic analysis of 3-D images for pathologists is outlined including the underlying methods of 3-D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer-aided analysis of large 3-D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3-D data is not in sight. A semi-automatic segmentation method is thus presented here.

Humans↗
PubMed1990

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.

Densitometry↗
PubMed1998

Comparison of high performance liquid chromatography and stereological image analysis for the quantitation of eumelanins and pheomelanins in melanoma cells.

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.

Chromatography, High Pressure Liquid↗
PubMed1999

Evaluation of a standardised procedure to assess the shape of pellets using image analysis.

The influence of threshold definition, number of pellets counted, image magnification and lightning technique on the assessment of pellet shape has been investigated using three batches of pellets and an image analysis system. The pellet parameters measured were 'aspect ratio', 'circularity', 'projection sphericity', 'e(R)' and 'Feret diameter.' The methodical error, reproducibility and repeatability of the results were chosen as statistical test parameters. The position of the light source is crucial in providing an accurate particle size value. Top light was identified as the illumination technique that gave a mean pellet size similar to the true pellet size. The use of a light table produced significantly larger pellet size values. A minimum pixel resolution appears necessary for an accurate shape parameter definition. One pixel should not cover more than 30 microm for pellets of an average particle size of 1.2 mm. Shape descriptors, which are based on a multiple combination of area and perimeter data such as the circularity, are greatly dependent on the number of pellets counted. Shape factors, which do not (aspect ratio) or only as a single value do involve an area or perimeter measurement (e(R), projection sphericity) are, however, nearly independent of the number of pellets counted, as long as the magnification is sufficiently large and the pellets are randomly drawn from the batch. For nearly spherical particles, the methodical error is below 1%, but for elongated particles this error can reach 5%. The repeatability is also very good for nearly spherical particles (<2%), but increases to very large values if the particles are clearly elongated. The limiting values for the various shape factors should be reconsidered. An upper value for the aspect ratio of 1.1 and a lower value of 0.6 for e(R) are recommended. The circularity should not be used as the shape factor to characterise spheres, because errors in image recognition can affect strongly the applicability of this shape factor. The projection sphericity has only a limited sensitivity to variations in particle shape.

Chemistry, Pharmaceutical↗
PubMedDate not supplied

In vitro assessment of incipient approximal carious lesions using computer-assisted densitometric image analysis.

OBJECTIVES: To investigate the use of computer-assisted densitometric image analysis (CADIA) to detect incipient approximal carious lesions in vitro and to compare areas of demineralization and mechanically induced lesions by radiographic visual inspection. METHODS: Thirty-six extracted non-carious human molars were demineralized in a defined approximal area of interest (AOI) using a gel system (pH 4.8) applied over 15 weeks. Standardized digital radiographs were taken before and after creation of caries-like lesions and grayscale values were measured within the AOI. For another group of eight teeth, artificial defects were mechanically induced by a diamond bur and densitometric analysis was performed. Statistical significance was tested using the Wilcoxon test and set at p < or = 0.05. In addition, radiographs were interpreted by visual examination by six experienced dentists. The sensitivity and specificity for both study groups were determined in comparison to a baseline group of untreated teeth. Polarized light microscopy and SEM analysis were used to evaluate changes in the subsurface mineralization pattern and surface micromorphology. RESULTS: Densitometric comparison of images from identical regions in control teeth revealed no significant grayscale value differences within the AOI, whereas a significant decrease (p < or = 0.05) was observed in the demineralized group. Mechanically induced lesions also showed a significant densitometric decrease (p < or = 0.05). The sensitivity of visual inspection of radiographs of demineralized areas was 0%. In contrast, the sensitivity for the detection of a mechanical defect of 0.1mm, which was the same subsurface extension as the histologically determined demineralization, increased to 52%. Specificity was 100% for all groups. CONCLUSIONS: CADIA is able to detect very small changes in mineralization of incipient carious approximal lesions in vitro. The findings presented in this investigation may improve the subtraction method used for diagnosis and monitoring of approximal caries, as well as prophylactic measures.

Absorptiometry, Photon↗
PubMed1977

[Investigations about ascertainment of exact morphological indices for the functionally activity of islets cells by means of electron optical characterizing and succeeded automatically image analysis of metal-containing secreting granules (author's transl)].

Biological image analysis with the "Quantimet 720" has been surrounded to date by difficulties due to the complexity of the image itself, and the difficulty of the Quantimet to discriminate between different objects on the basis of their grey levels alone (Bradbury 1975). Although the makers of image analysing computers try to diminish these difficulties by contructing new models of computers in future, we have the opinion, that it is possible to receive already today excellent results in image analysing of structures in biology and medicine under the following condition: selective staining of objects, we want to measure by simultaneous discriminating of those structures, which influence negatively the image analysing on the basis of their grey levels.

Animals↗
PubMed1996

Enzyme-linked immunosorbent assay by image analysis using a charge-coupled device array detector.

This paper describes a fluorescence enzyme-linked immunosorbent assay (ELISA) for the quantification of (+/-)-2-(2, 4-dichlorophen-oxy)propionic methyl ester (dichlorprop methyl ester). Antibodies for dichlorprop methyl ester were produced by immunizing rabbits with a conjugate of dichlorprop methyl ester with bovine serum albumin. Data acquisition on microtiter wells is performed by a spectrofluorometer through a fiber optic and by a charge-coupled device camera. A correlation was obtained between the image analysis data on ELISA and the data acquired by the spectrofluorometer. The results demonstrate that the fluorescence image analysis performed by the charge-coupled device detector is applicable to ELISA, and the analysis time, sensitivity, and precision of the ELISA procedure are compared to conventional fluorescence ELISA performed by the spectrofluorometer. The ELISA procedure was selective for structurally similar compounds or usually found in formulation pesticides. Concentrations for 50% displacement curves were dichlorprop, 83.59 microg/ml, and 2,4,5-T, 388.23 microg/ml; triclopyr, ioxynil, bentazone, and MCPA had no response.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

Aneugen-induced micronuclei (MN) in human lymphocytes may be discerned using image analysis techniques when cell-cycle stage is taken into account.

We show that for the in vitro cytochalasin-B human lymphocyte micronucleus (MN) test, the quantification of the DNA content of MN and the difference in DNA content between the two macronuclei in the binucleate cells without MN, as measured by image analysis, gives a first estimation of the aneugenic potential of a test compound. Cultures of isolated human lymphocytes were exposed either to gamma-rays as a clastogen or to carbendazim (MBC) as an aneugen. The lymphocytes were stained with Feulgen stain and the MN were analyzed for DNA content with a Magiscan 2A image analyzer. The mean DNA content of MN induced by MBC were statistically higher than gamma-irradiation-induced MN. It was demonstrated that in culture the lymphocytes, as well as the MN, are in different stages of the cell cycle, but this will not affect the discriminating power of the MN DNA content when only G1 cells are considered, or when DNA content of the MN is expressed relative to the total genome. The identification of G1 and G2 cell populations from image analysis data was performed by extrapolation of DNA content data from G1- and G2-sorted lymphocytes with a FacStar plus flow sorter. It was demonstrated that in MBC-treated cells the DNA rearrangement between the macronuclei in binucleates without MN was on the average higher than in gamma-irradiated and untreated cells, which points to aneugenic effects of MBC without the formation of MN. In contrast to DNA content measurements, the area of the MN is not a reliable measure for discriminating clastogens from aneugens.

Aneuploidy↗