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Potential use of spectral image analysis for the quantitative evaluation of estrogen receptors in breast cancer.

Evaluation of estrogen receptor (ER) content is an important factor in the choice of therapy and prognosis of breast cancer patients. In this study, we demonstrate a new spectral image analysis technique for objective and quantitative evaluations of stained specimens. The SpectraCube system was used to analyze nuclear antigens in thirteen cases of breast cancer stained by the immunoperoxidase method with hematoxylin counterstain. Spectral imaging segregated the spectrum of diaminobenzidine (DAB) from the background color of hematoxylin and a spectral index was calculated. The spectral index essentially agreed with the pathologist's index (on a scale of 0 to 3) in seven out of the thirteen cases. A substantial number of ER positive pixels was detected in the two cases scored as 0 by the pathologist's index. In a test case scored as 1 by the pathologist's index we detected a significant number of pixels, representing 47% of the nuclei, with DAB-intensity values higher than the cut-off value of 1.2. These data suggest that spectral image analysis is a sensitive method providing intensive information with high reproducibility. Our spectral imaging method is highly flexible, enabling the user to define the spatial resolution of the analyzed specimen by choosing the number of pixels per one nucleus.

3,3'-Diaminobenzidine↗

Microelectrode-guided posteroventral pallidotomy for treatment of Parkinson's disease: postoperative magnetic resonance imaging analysis.

The authors report the postoperative magnetic resonance (MR) imaging findings in 36 patients with advanced Parkinson's disease who underwent unilateral microelectrode-guided posteroventral pallidotomy. The lesions were placed within 1 mm of the ventral border of the globus pallidus internus (GPi) to include pallidothalamic outflow pathways. Sequential MR studies were obtained within 1 to 3 days postoperatively and at 6-month follow-up examination. Thirty-four (94%) of the 36 patients enjoyed sustained moderate or marked improvement of their parkinsonian symptoms 6 months postoperatively. Transient side effects occurred in five patients (14%), but there were no persistent complications. The pallidal radiofrequency lesions were prolate spheroid shaped and were composed of three concentric zones in the early postoperative studies. The mean volume of the middle zone, corresponding to the area of hemorrhagic coagulation necrosis, was 44.4 +/- 17.6 mm3; the mean lesion volume as defined by the outer zone, corresponding to perilesional edema, was 262.2 +/- 111.6 mm3. Additional edema spreading to the internal capsule was noted in 32 of 34 cases and to the optic tract in 11 of 34 cases. In two patients small ischemic infarctions involving the corona radiata were found, and in one a venous infarction was detected. Ischemic infarction resulted in mild transient Broca's aphasia in one patient, but there was no detectable neurological deficit in the other two. The mean volume of late-phase (6 months) lesions was 22 +/- 28.8 mm3. In three patients no lesion was identified despite sustained clinical improvement. The lesion was located in the posteroventral GPi in all cases except in one patient in whom it was confined to the GP externus (GPe). This 49-year-old woman did not experience sustained benefit. The authors found no consistent correlations between lesion size and location and clinical outcome as measured by a global outcome score, the Unified Parkinson's Disease Rating Scale motor, activities of daily living, and bradykinesia "off" scores or rating of dyskinesias. Lesioning of pallidal and subpallidal pathways may contribute to the sustained clinical benefit in this series. Magnetic resonance imaging analysis showed that intraoperative microelectrode recording facilitated accurate placement of the lesion in this critical area.

Adult↗

Predicting the onset of filamentous bulking in biological wastewater treatment systems by exploiting image analysis information.

The performance of the activated sludge process is limited by the ability of the sedimentation tank (1) to separate the activated sludge from the treated effluent and (2) to concentrate it. Apart from bad operating strategies or poorly designed clarifiers, settling failures can mainly be attributed to filamentous bulking. Image analysis is a promising technique that can be used for early detection of filamentous bulking. The aim of this paper is therefore twofold. Foremost, correlations are sought between image analysis information (i.e., the total filament length per image, the mean form factor, the mean equivalent floc diameter, the mean floc roundness and the mean floc reduced radius of gyration) and classical measurements (i.e., the Sludge Volume Index (SVI)). Secondly, this information is both explored and exploited in order to identify dynamic ARX and state space-type models. Their performance is compared based on two criteria.

Algorithms↗

Statistical characterization of morphological features of layer-by-layer polymer films by image analysis.

This article presents an image analysis-based approach to the statistical characterization of morphological features in layer-by-layer (LBL) polymer films, which depend on the experimental conditions of fabrication. The method is based on the recently introduced concept of exact dilations. This concept allows the precise determination of all possible parallel contours around any general object and, as a consequence, the respective fully accurate Voronoi partition defined by the objects. With the use of a fast wavefront propagation scheme, such diagrams can be obtained at high speed. The method has been applied to statistically characterize features such as grain size and distribution on LBL polymer films. To do this, the polymer surface is tracked from its highest z values downward to its bottom, and each new connected component is detected as a Voronoi seed. Through determination of equivalent radii of the Voronoi cells, the probability density function characterizing the distribution of the domain radius can then be obtained.

Image Processing, Computer-Assisted↗

On-line size measurement of yeast aggregates using image analysis.

Alcohol fermentation productivity can be strongly improved using a flocculation-based yeast recycle. However, the efficiency of the biomass retention system depends strongly on the yeast particle size. Accordingly, the monitoring and control of yeast floc diameter are of primary importance. The on-line measurement of mean floc diameter has been achieved using on-line image analysis, based on the evaluation of image texture. The texture analysis method consisted in the building of a co-occurrence matrix from which the so-called "Energy parameter" was extracted. While image texture is usually used for classification purposes, it has been used here as a quantitative descriptor: a correlation has been found between this statistical image feature and off-line manual floc-size determinations. In the floc-size range investigated (X 0.5-4.3 mm), the evaluated mean diameter was in good agreement with the actual particle size, with a determination coefficient equal to 0.980. In contrast with manual measurements, slow and tedious, this method gave the value of the mean particle diameter in real-time, without sampling. This novel tool has been used to investigate the behavior of yeast aggregates as a function of fermentation conditions. While biomass concentration was kept constant, step increases of the feed rate led to a decrease of the mean floc diameter. Image analysis showed that the particle-size reduction could occur within a few minutes after modification of the medium dilution rate, demonstrating the disruptive effect of the CO(2) efflux. The kinetic of aggregate formation was dependent on the gas-phase composition. Instead of recycling fermentation gas, sparging the fermentor with nitrogen, to reduce dissolved CO(2) concentration, increased the rate of floc-size growth.

Automation↗

An image analysis technique for the investigation of variations in placental morphology in pregnancies complicated by preeclampsia with and without intrauterine growth restriction.

OBJECTIVE: The purpose of this study was to use visual image analysis to observe changes in the morphology and composition of placental villi in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). METHODS: Placental biopsies from nine normal pregnancies, five cases of PE, five cases of IUGR, and five cases of PE with IUGR (PE x IUGR) were randomly sampled. Formalin-fixed, wax-embedded sections were stained with hematoxylin and eosin (H&E) and subjected to image analysis. The placental areas occupied by villi, syncytiotrophoblast, and syncytial cytoplasm and nuclei were quantified. RESULTS: Significantly smaller placentas were obtained from growth-restricted pregnancies. PE, with and without IUGR, had no effect on the total area occupied by villi or intervillous space. IUGR alone showed a real and consistent reduction in villous area (56.0 +/- 2.4% vs 43.6 +/- 3.3%, P <.03). While the ratio of syncytial to villous areas were noticeably reduced in all cases of PE (0.38 +/- 0.03 vs 0.24 +/- 0.07, P <.05), this ratio remained unchanging in IUGR. Birth weight was positively correlated to both placental size and total villous area occupied. Moreover, increasingly positive relationships were recorded between both syncytiotrophoblast area and syncytiotrophoblast cytoplasm and birth weight (P <.01 and P <.001, respectively). CONCLUSION: These measurements point to impoverished villus development in idiopathic IUGR. The observed changes in PE with IUGR were more akin to PE without growth restriction than IUGR alone. This suggests that idiopathic IUGR and IUGR in PE have a separate etiology, idiopathic IUGR arising through a reduction in villous area alone, and IUGR in PE caused by changes in syncytiotrophoblast quantity, more specifically the amount of syncytiotrophoblast cytoplasm.

Adult↗

Two-color image analysis discriminates between mineralized and unmineralized bone nodules in vitro.

Functional assays of progenitor cell capacity for colony formation in vitro typically depend on the investigator's expertise with quantification. The ability to enumerate and analyze colony types with standardized criteria with no bias would be a useful tool for research and drug development. We report the development of a two-color automated analysis system for colony-forming unit-osteoblasts that is capable of reporting progenitor frequency and bone nodule number size, and type (mineralized or unmineralized). Our image analysis system was validated using the rat calvaria cell model to measure in vitro bone nodule development. With computer-aided image analysis, data on nodules can be rapidly generated with a minimum of user bias and fatigue. This novel tool will distinguish mineralized and unmineralized bone nodules, facilitates quantification, enable large-scale experimental design, allow for long-term data storage and tracking, and lead to the identification of new parameters that impact bone development.

Animals↗

A quantitative method to measure alkaline phosphatase activities in individual leukocytes by image analysis.

Leucocyte alkaline phosphatase (L-ALP) is well known as leukemia marker, but recent results suggest its usefulness for the diagnosis of several diseases. The aim of this study was to develop a quantitative method to measure alkaline phosphatase activities in individual leukocytes by image analysis. We studied the reaction rate of L-ALP in human polymorphonuclear leucocytes by a microscope attached to a TV camera and a computerized image analyzer. The optical density (OD) measured was standardized by grey filters with known absorbance. We measured IOD for individual cells after a set incubation time by end-point measurements. Studies of kinetic parameters of L-ALP were performed by single-point measurements in the linear phase of the reaction and at increasing substrate concentrations. Cellular IOD increased proportionally with incubation time up to 10 min. The mean KM(mM) and Vmax(delta IOD/min) values were 0.70 +/- 0.11 and 1.76 +/- 0.2 (mean +/- SE, n = 5) respectively. Our findings are comparable to previous results using a polyvynil alcohol method in microphotometry analysis. The image analysis of cellular L-ALP activity appears a valuable tool for quantitative studies.

Alkaline Phosphatase↗

Quantization of widefield fluorescence images using structured illumination and image analysis software.

It is difficult to obtain precise quantitative measurements from fluorescent images captured from widefield microscopes. We wished to ascertain if reliable quantitative measurements of both biological and nonbiological specimens were possible using a widefield microscope equipped with a structured illumination system and image analysis software. In a nonbiological specimen, images were obtained from fluorescent beads of known intensity. For a biologically relevant model we used the uptake of fluorescently labeled transferrin by HeLa cells in culture. In the bead sample, the mean intensity of beads acquired with the structured illumination system showed relative intensities near to the predicted values without any further manipulation of data. In the transferrin uptake experiments, uptake and subsequent recycling of the labeled transferrin could be accurately and reproducibly measured. We conclude that using a combination of structured illumination and image processing algorithms accurate quantization of fluorescent images can be achieved in widefield microscopy.

Algorithms↗

The prognostic value of image analysis in ovarian cancer.

Histologic grading is very important for treatment decisions in ovarian cancer. All grading systems contain a significant subjective component, which could be reduced by including objective measurements into the diagnostic decision. Image analysis was used to determine nuclear area and ploidy distributions in 42 patients with epithelial ovarian cancer, and the results were related to tumor grade and clinical outcome. The mean nuclear area, mean optical density, number of hyperploid cells, and the standard deviation between measurements were significantly higher in Grade 2 and 3 tumors compared with Grade 1 tumors, in rapidly progressive tumors compared with less aggressive malignancies, and in recurrent tumors compared with primary lesions. The number of nuclei with very high DNA content was found to be of prognostic importance. Image analysis thus provides additional prognostic information in epithelial ovarian cancer.

DNA, Neoplasm↗

Quantitative study of KI-67 antibody staining in non-Hodgkin's lymphomas using image analysis.

In sections from 32 B malignant lymphomas (ML), the total KI-67 stained area was compared to the number of KI-67 positive cells in order to demonstrate the reliability of using image analysis to quantify the proliferative activity. The total KI-67 area percentage correlated highly with the number of KI-67 positive cellular profiles (r = .93). Significant differences were found between low- and high-grade ML according to the Kiel classification (mean values +/- SD, respectively, of 7.7 +/- 3.81% and 16.6 +/- 6.23%), and between low-, or intermediate- and high-grade ML only, according to the International Working Formulation. Within the Working Formulation, the statistical analysis grouped the diffuse large cell subtype of intermediate grade with the immunoblastic high-grade subtype. A wide range of KI-67 area percentage values was noted, particularly in follicular ML; for these follicular ML, considering follicular areas only, values were comparable to high-grade ML (14.8 +/- 6.60%). In conclusion, the KI-67 area percentage is a reliable alternative method to manual cell counting, and image analysis allows quicker measurements appropriate to large and strictly lymphomatous areas, using a greater number of cells than in manual cell counting.

Antibodies, Monoclonal↗

The effect of culture conditions on the morphology of the dimorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415: a study incorporating image analysis.

The effect of changing environmental conditions on the morphology of the yeast Kluyveromyces marxianus var. marxianus NRRLy2415 was investigated in batch and continuous culture, using a previously developed computer-aided image analysis protocol [O'Shea and Walsh (1996) Biotechnol Bioeng 51: 679 690]. The morphology of the organism is primarily controlled by the specific growth rate, mu. This finding was contrary to a previous investigation [Walker and O'Neill (1990) J Chem Tech Biotechnol 49: 75-89]. When the organism is cultured in batch with excess oxygen, mu can approach the maximum specific growth rate, microm, and the primary morphology of the culture is yeast-like. However, if the organism is cultured in a chemostat, thereby controlling the growth rate, the morphology reverts to a pseudohyphal form. This response is thought to be an adaptation by the organism to its environment, whereby it assumes a foraging form under adverse environmental conditions. The use of computer-aided image analysis made possible the discrimination of subtle morphological differences between samples and the determination of the relationship between morphology and growth rate.

Biomass↗

[Measurement of nucleolar organizers (AgNOR) using the LUCIA-M color image analysis system].

Argyrophil Nucleolar Organizer Regions (AgNORs) reflect the ploidy and proliferative activity of cells. In a Czech made system of image analysis LUCIA-M (Laboratory Universal Computer Image Analysis-Medium version) we have created a macro for AgNORs evaluation. The process includes picture contrasting and processing, thresholding of nuclei and AgNORs, elimination of artifacts through restrictions and interactive editation. The relation of nuclei to AgNORs is realized using binary operations. The number of dots in a nucleus is counted using advanced morphology-homotopic marking. The object data (nuclear area, absolute and relative AgNOR area, mean equal diameter of nuclei, circularity) may be processed either in the system LUCIA itself or exporting the data into another statistic program, e.g. EXCEL. The choice of the most informative features measured for diagnostics and prognostics of tumours is a task for subsequent clinico-morphologic studies.

Animals↗

An alternative method for electrophoretic gel image analysis in the GelMaster software.

A novel methodology of electrophoretic gel image analysis has been proposed that is based on two-dimensional image processing methods instead of previously used one-dimensional Gaussian deconvolution. The crucial problem of the analysis of imperfect gels, that consists in band detection, is solved using the algorithms of band boundary detection and intensity homogeneity indication. The template approach represents the core element of the developed algorithms. The GelMaster software system has been developed in which the novel algorithms are implemented. It involves two-stage interaction with the user: detection of the true bands and deleting the false band detections. The main features of the GelMaster system and the most important algorithms are described.

Algorithms↗

Progress in the measurement of ciliary beat frequency by automated image analysis: application to mammalian tracheal epithelium.

The investigations on the activity of respiratory ciliae in vivo are of considerable interest for the study of physiopathological disorders resulting from chronic infection or inhaled pollutants. We present an image analysis method for the measurement of ciliary beat frequency on mammal or human in vivo samples. Videofilm sequences of samples placed under the microscope were analyzed frame by frame by image analysis. The operator defines on the screen several areas of interest on the cell apex. The variation of grey levels in each areas allows the calculation of ciliary beat parameters. This method permits one to investigate the effects of pharmacological agents or noxious pollutants on ciliae beating and the physiological causes underlying them. Our results on rabbits were in agreement with the data published in literature, showing no significant difference between intratracheal frequencies obtained at medium and lower levels. The upper level near the cracoid cartilage has shown a greater variability than the other two levels with higher beating frequencies, a difference of likely physiological importance.

Animals↗

Simultaneous multiple site arteriolar vasomotion measurement using digital image analysis.

An automatic digital image processing technique for simultaneous vasomotion analysis in peripheral microcirculation at multiple sites and in real time is presented. The algorithm utilizes either fluorescent or bright field micro-images of the vasculature as input. The video images are digitized and analyzed on-line by an IBM RT PC, using digital filtering and edge detection. The sampling frequency is higher than 5 Hz when only one site is tracked and decreases as the number of sites is incremented. Performance of the algorithm was tested for a hamster cutaneous microcirculation model.

Algorithms↗

How echographic image analysis of venous oedema reveals the benefits of leg elevation.

OBJECTIVE: Venous insufficiency, leading to venous oedema, is a key pathogenic factor for the non-healing of venous leg ulcers. This study aimed to evaluate the effects of leg elevation on venous oedema. METHOD: Ten patients aged 44-89 years (median: 61) with leg oedema had high-frequency B-mode ultrasound scanning and digital image analysis before and after three to four hours of leg elevation. The echographic image analysis system was used, where oedema is represented by the hypoechogenic part of the image--that is, the total number or density of low echogenic pixels (LEPs) in a particular area. RESULTS: Compared with pre-elevation, the volume of the lower leg decreased by 2.9% +/- 0.6 (138 cm3 +/- 39) after three to four hours' elevation (p < 0.05). After elevation, the LEPs in the upper, middle and lower sites of the limb decreased by 8.8%, 15.6% and 17.3% respectively, reaching statistical significance (p < 0.05) in the lower site. The ratio of LEPs in the upper and lower dermis in the upper, middle and lower sites decreased by 30.3%, 45.8% and 22.5% respectively. This was significant in both the middle and lower sites (p < 0.01). After elevation dermal thickness increased by 0.047 mm, 0.194 mm and 0.232 mm respectively. This change was statistically significant in the middle (p < 0.05) and lower sites of the limb (p < 0.01). CONCLUSION: LEPs are a sensitive marker of dermal oedema and its effects. Leg elevation is extremely effective in reducing oedema, even if only for three to four hours.

Adult↗

Classification and counting of fluorescent pollen using an image analysis system.

Pollen viability is commonly assessed by fluorochromatic reaction (FCR) because of the high correlation between positive fluorescence of the pollen grains and their ability to germinate. One of the advantages of this method is its simplicity. An experiment to test FCR analysis for reproducibility, however, showed that results are affected by subjectivity. There is little consistency between analysts, and assessment by the same analyst may differ for the same pollen sample image examined at different times. These problems were solved by a computerized image analysis system that provides a method for classifying positive and negative fluorescent pollen and automatic counting of the grains in each class. The computerized image analysis system does not change the biochemistry of the FCR test, but avoids some experimental errors owing to the subjectivity of the analyst. Microscope images of the pollen after FCR were digitized and later analyzed by specially designed software, "Plant Meter." This software deletes the dark background of the image to isolate the grains, and subsequently counts positive and negative fluorescent pollen grains. An experiment was carried out to validate software output and it showed reliable results. Moreover, the software is user friendly and very little training is necessary for analysts to achieve reliable results.

Cell Count↗