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Skeletal muscle tissue preparation for image analysis systems.

A procedure for making histological sections of whole skeletal muscles suitable for automated image analysis of fiber size and number is described. Briefly the procedure involves the fixation/embedding technique of Schmitt and Dumont (1969) followed by section staining using phosphomolybdic acid and aniline blue. This procedure overcomes many of problems encountered with the more usual muscle section techniques in that it suppresses muscle fiber staining and stains only the intramuscular connective tissue.

Aniline Compounds↗

Measuring of AgNORs using image analysis.

A method is presented for the measurement of AgNORs in histological section using an image analysis system. Measurements are performed at the highest magnification (objective 100x) using a CCD camera. In the first step the AgNORs are segmented, counted and measured. In the second step the AgNOR clusters are segmented. Several features for the description of the AgNORs, the AgNOR clusters and their spatial distribution throughout the nucleus are given. As an internal reference, lymphocytes are used and the size features are given relative to the mean size of AgNORs in the lymphocytes. The reproducibility of the features was tested on a series of astrocytomas. This should be carried out with at least 150 tumor cells and 20 reference cells in order for accurate measurements.

Astrocytoma↗

Image analysis of fundus photographs. The detection and measurement of exudates associated with diabetic retinopathy.

A computer-based image analysis system was used to detect and measure exudates in fundus photographs. A fundus transparency was imaged, digitized, and stored in image memory. The stored image was then processed by several operators, to reduce shade variations in the image background and enhance the contrast between this background and the exudates. Exudates were separated from the background on the basis of their brightness or "gray level" and were then copied in to a binary image. For comparative purposes, the binary image was superimposed on the original unprocessed image. Exudate areas were measured using the binary image, which was also transferred to a printer to provide a permanent record or "exudate map." The system was able to discriminate between standard photographs used to grade hard exudates in the Early Treatment for Diabetic Retinopathy Study (ETDRS). It was also used to monitor the response of a subject to laser treatment.

Diabetic Retinopathy↗

Image analysis techniques for automatic evaluation of two-dimensional electrophoresis.

Techniques for automatic analysis of two-dimensional electrophoresis gels by computer-aided image analysis are described. Original gels or photographic films are scanned using a laser scanner and the files are transferred to a microcomputer. The program package first performs a compression and preevaluation of the files. Spot identification and quantification is performed by the chain code algorithm after appropriate zooming and cutting. Labeling facilitates spot identification and quantification in numerical and graphical (pseudocolor) representation on peripheral devices for camera ready output. Interpolation between measured basepoints is performed by cubic spline algorithms which are automatically switched on and off, depending on the need by the program. High speed analysis and graphic representation is achieved using fast Assembler language routines rather than high level languages. One-dimensional gels can be analyzed using the same software. Spot matching between parallel two-dimensional gels has not yet been implemented.

Electrophoresis, Gel, Two-Dimensional↗

[A quantitative study on DNA content of salivary gland carcinomas by image analysis technique].

DNA content was quantitatively determined in 30 cases of salivary gland carcinoma (SGC) by image analysis technique using paraffin-embedded sections stained with Feulgen method. The results showed that the nuclear DNA content was related to the malignancy of SGC, based on the clinical and morphological criteria such as tumor size, mobility, property of growth, mitotic rate, degree of differentiation and the histological type of the tumor. The number of nuclei above 5 C DNA, expressing aneuploidy, seems to be of more prognostic significance. Aneuploidy greater than 10% may be indicative of a poor prognosis for SGC. Thus, it is suggested that the quantitative analysis of DNA content may be able to provide an objective and quantitative reference for evaluating the prognosis of SGC.

Adenocarcinoma↗

Image analysis of the axonal ingrowth into poly(D,L-lactide) porous scaffolds in relation to the 3-D porous structure.

Porous polymer scaffolds are promising materials for neural tissue engineering because they offer valuable three-dimensional (3-D) supports for the in vitro and in vivo axonal growth and tissue expansion. At the time being, how the in vivo neuronal cell development depends on the scaffold 3-D architecture is unknown. Therefore, scanning electron micrographs of longitudinal sections of porous polylactide scaffolds and immunohistological sections of these scaffolds after implantation and neurofilament staining have been studied by image analysis. Pore orientation and axonal ingrowth have been investigated by spectral analysis on gray level SEM images. Binary image processing has been carried out and the binary images have been studied by spectral analysis in order to estimate the possible effect of the image noise on the real pattern. In addition to axonal orientation, density and length distribution of the regenerated axons into the polymer scaffold have been measured. Dependence of the axonal ingrowth on the 3D-polymer scaffold has been discussed on the basis of the collected data.

Absorbable Implants↗

Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy.

To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light microscope was used. Retardation values of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage, the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predominantly from the intrinsic (structural) birefringence. In conclusion, analysis of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid quantitative assessment of oriented collagen structures in articular cartilage.

Animals↗

Applications of quantitative digital image analysis to breast cancer research.

Our studies of radiogenic carcinogenesis in mouse and human models of breast cancer are based on the view that cell phenotype, microenvironment composition, communication between cells and within the microenvironment are important factors in the development of breast cancer. This is complicated in the mammary gland by its postnatal development, cyclic evolution via pregnancy and involution, and dynamic remodeling of epithelial-stromal interactions, all of which contribute to breast cancer susceptibility. Microscopy is the tool of choice to examine cells in context. Specific features can be defined using probes, antibodies, immunofluorescence, and image analysis to measure protein distribution, cell composition, and genomic instability in human and mouse models of breast cancer. We discuss the integration of image acquisition, analysis, and annotation to efficiently analyze large amounts of image data. In the future, cell and tissue image-based studies will be facilitated by a bioinformatics strategy that generates multidimensional databases of quantitative information derived from molecular, immunological, and morphological probes at multiple resolutions. This approach will facilitate the construction of an in vivo phenotype database necessary for understanding when, where, and how normal cells become cancer.

Animals↗

Quantitative image analysis of the chromatolysis in rat facial and hypoglossal motoneurons following axotomy with and without reinnervation.

Image analysis was used to quantify the time course of chromatolysis in regenerating and degenerating motoneurons. Following facial-facial, hypoglossal-hypoglossal nerve suture, or resection of facial and hypoglossal nerves with postoperative survival times of 4 h to 112 days, the texture of the Nissl substance of facial and hypoglossal motoneurons was analyzed on both sides of the brainstem in paraffin serial sections with a VIDASplus image analyzer. In this quantitative study of 149 Wistar rats, alterations of the Nissl substance were measured that were statistically significant but not yet visible to the human eye. Chromatolysis started significantly as early as 8 h and was not fully reversed 112 days after any of the types of axotomy. The reaction was more intense and longer lasting following axotomy without reinnervation than with reinnervation. Thus, chromatolysis starts much faster and lasts far longer than was previously known. The quantified chromatolysis is much stronger after permanent target deprivation than during complete regeneration of motoneurons but is reversible in both cases.

Animals↗

Quantitative nuclear morphometry, Markovian texture descriptors, and DNA content captured on a CAS-200 Image analysis system, combined with PCNA and HER-2/neu immunohistochemistry for prediction of prostate cancer progression.

One hundred and twenty-four localized prostate cancer patients operated on at Johns Hopkins Hospital (JHH) since 1975 were identified. The sample was optimized for evaluation of prostate cancer progression. Based upon accurate clinical histories, these radical prostatectomy patients included 50 progressors and 74 non-progressors using appearance of serum PSA as an indication of recurrence (mean follow-up = 8.6 +/- 1.8 years, range 7-15 years). All patients included in the study had no involvement of their seminal vesicles or lymph nodes at the time of prostatectomy. Average time to progression was 3.6 +/- 2 years, range of 1-8 years. Using paraffin-embedded specimens, several five micron sections were cut and placed on Probe-On slides; one slide was H&E-stained and the other was Feulgen-stained. The H&E and Feulgen-stained slides were screened and "dotted" by pathologists at JHH and CytoDynostics, Inc. A CAS-200 Image analysis system (Cell Image Systems, Elmhurst, IL) equipped with a Cell Measurement Program version 1.2 beta, was used to capture the Feulgen-stained images and to perform the calculations. From the "dotted" areas, 150 cancer cells were selected for measurement of DNA content and 27 nuclear morphometric shape and size factors, including 21 Markovian chromatin texture variables. Additional sections were used for immunochemistry staining with an alkaline phosphatase streptavidin-biotin complex stain to detect and quantitate cancer cells binding monoclonal antibodies directed against proliferating cell nuclear antigen (PCNA) and HER-2/neu antigen. All data were entered into a statistical program (STATA) for further analysis and univariate and multivariate statistical analysis was performed using logistic regression and its stepwise variant. The biomarkers of greatest utility to detect progressors when analyzed univariately included post-operative Gleason score (p = < 0.0001), HER-2/neu antigenicity (p = 0.0147), CAS-200 DNA ploidy (p = 0.008), and twelve Markovian nuclear texture and shape features (p = < 0.0001), whereas PCNA (p = 0.160) failed. The optimal set of nuclear morphometry progression tumor features were selected using backward stepwise logistic regression estimate analysis which drops variables due to collinearity. Although post-operative Gleason score is a strong univariate predictor of progression, DNA ploidy and HER-2/neu contributed significantly to further stratification of higher risk groups within the low Gleason score subpopulation. The best Markovian features combined with post-operative Gleason score generated sensitivity = 90%, specificity = 96%, positive predictive value = 94%, negative predictive value = 93% and the area under the receiver operator curve was 0.975.

Biomarkers, Tumor↗

Sperm morphology by image analysis compared with subjective assessment.

Human sperm heads were detected and tracked using semi-automatic image analysis. Six measurements of size and shape were computed from two samples of semen from each of 26 men. Subjective assessments of abnormal morphological forms were made on the same samples by two independent assessors. A formula for prediction of percentage abnormality was calculated from the results. The subjective assessment of abnormality was related primarily to the number of elongated heads and secondly to the number of small heads. Computed image processing provides an objective method for measurement of the morphological features of sperm.

Humans↗

Techniques of image analysis for quantitative immunohistochemistry.

The aim of this paper was to evaluate the usefulness of digital image analysis techniques to measure the amount and strength of immunohistochemical markers. The new method, based on the spatial visualization technique, was confronted with methods of colour sampling and grey scale thresholding. Examples of applications of the techniques for apoptosis and proliferation markers are also presented.

Biomarkers↗

Detection and measurement of approximal radiolucencies by computer-aided image analysis.

Serial standardized bitewing radiography permits monitoring of the development of approximal carious lesions. A quantitative method using image-analysis techniques was developed. Only certain types of hardware proved capable of processing this complex image adequately. A computer-aided, software-driven, TV-based system incorporating an image memory, recursive filtration, and 256 gray-level resolution, which can both detect and measure approximal enamel radiolucencies produced by natural carious lesions, has been developed. The radiolucent boundary is delineated, displayed, and plotted to scale. For the first time, objective values for depth, area, width, perimeter, and "degree of radiolucency" can be automatically derived. Currently the system is seen as a tool in caries research; in the future such systems may have wider applications.

Computers↗

Expression of E2F-4 gene in colorectal adenocarcinoma and corresponding covering mucosa: an immunohistochemistry, image analysis, and immunoblot study.

E2F-4 is a transcription factor involved in the transition of the cell from the resting state (G0/G1) to the proliferative stage (S). It has been associated with the p107 and p130 members of the Rb-family and it is responsible for many important growth suppressive functions. E2F-1, one member of the E2F family, has a similar structure to E2F-4; however, both have different mechanisms of action in regulating cell-cycle progression. Although E2F-4 acts mainly as a repressor in the early part of the cell cycle, E2F-1 has the ability to function as both an oncogene and a tumor suppressor gene. In an attempt to identify the role of E2F-4 as a potential mediator of cell proliferation, differentiation, tumorigenesis, and apoptosis in colorectal mucosa comparing with that of E2F-1, the authors examine 20 patients with human colon cancer and their corresponding histologically healthy mucosa by using immunohistochemical methods, computerized quantitative image analysis, and immunoblot analysis. Immunohistochemical studies were performed with formalin-fixed, paraffin-embedded sections stained with a monoclonal antibody against the E2F-4 protein. Apoptosis levels were determined by in situ assay. Positivity was scored by a Computerized Image Analyzer to detect the relative amount of the protein. Immunoblot analysis was performed on protein extracts from snap-frozen tissues of the same specimens. The results show that the expression of E2F-4 was greater in the tumor cells than in their corresponding benign epithelium as determined by immunohistochemical staining and image analysis. This was confirmed by semiquantitative IB analysis of the E2F-4 protein. The labeling index (LI) of E2F-4 in the tumors was inversely proportional to the LI of apoptotic cells. Within these cases, 12 cases showed a very high E2F-4 LI corresponding to low apoptosis LI. Three cases with relatively lower levels of E2F-4 LI were characterized with high apoptotic rates. These data suggest that E2F-4 gene overexpression plays a role in the development of colorectal tumors and appears to play a role in suppressing apoptosis.

Adenocarcinoma↗

Computer-based intraoral image analysis of the clinical plaque removing capacity of 3 manual toothbrushes.

BACKGROUND: (I) Introducing an intraoral camera system with a special positioner to allow computer-based analysis of reproducible images on lingual tooth surfaces and (II) comparing plaque removal by three manual toothbrushes with different brushhead designs (convex, multilevel and flat trimmed) on lingual mandibular tooth surfaces. METHOD: In a clinical single-blind, crossover, 24-h plaque-regrowth study on 25 subjects, a computer-based index (PPI) was used to evaluate pre- and postbrushing plaque on lingual surfaces of mandibular premolars and molars. Subjects brushed their teeth under standardized conditions at three visits, each time with a different, randomly assigned toothbrush. RESULTS: The intraoral camera system allowed a reproducible and relatively convenient access to the lingual surfaces of the mandibular teeth and provided an increase in objectivity. Overall, each brush achieved statistically significant plaque removal, however, none reached clinical relevance. The multilevel brush was superior at specific sites, but failed to show statistically significant superiority in terms of overall plaque reduction. Without regard of the toothbrush used, the right handed subjects were less efficient in removing plaque from the right side compared to the left. CONCLUSIONS: The method is able to detect even small differences in plaque reduction. None of the different brushhead designs was able to compensate an insufficient brushing techniques.

Adult↗

Protein determination by ponceau S using digital color image analysis of protein spots on nitrocellulose membranes.

A procedure was developed to estimate protein concentrations using color image analysis of protein spots stained with ponceau S. The method involved spotting a constant volume (2 microl) of the protein solutions on nitrocellulose paper, staining with acidic ponceau S, destaining, and air drying the paper. The image of the nitrocellulose paper was grabbed using a digital color scanner and thresholded with an optimal value to mark the area of the spot. The intensity of the color in the spot was measured in an arbitrary unit of intensity termed as inverse integrated gray value. This value showed a discernible increase with protein concentrations from 0.1 to 50 microg protein per spot (0.05-25 mg/ml). The method is simple and convenient compared to the conventional spectrophotometric procedures and allows several samples to be analyzed simultaneously. It can also be used to estimate protein concentration in the spots stained with Coomassie brilliant blue or other dyes.

Collodion↗

DNA ploidy of fibrolamellar hepatocellular carcinoma by image analysis.

Twelve cases of fibrolamellar hepatocellular carcinoma (FLC) were evaluated for DNA ploidy by means of image analysis of Feulgen-stained tissue sections. All of the tumors showed a nondiploid DNA distribution (six aneuploid and six tetraploid); no diploid pattern was found. The nuclear area of the tumors (53.8 microns 2 +/- 18.0; mean +/- standard deviation) was significantly larger than that of the surrounding noncancerous livers (33.2 +/- 4.7; P < .0001). These findings suggest that DNA content in fibrolamellar carcinoma (FLC) is not directly related to the clinical behavior and that other factors may be responsible for the better prognosis of this variant of HCC.

Adolescent↗

[Quantitative computerized image analysis of the relationship between neovascularization and nonperfusion area in branch retinal vein occlusion].

Quantitative computerized image analysis of composite fluorescein angiograms of 62 eyes with branch retinal vein occlusion (BRVO) found 33 eyes with newly formed blood vessels. Determination of the ratio of the retinal capillary nonperfusion area to the disk area revealed that the ratio was positively correlated with the incidence of neovascularization, in that 10.0% of the cases with the ratio under 10 and 63.2% of the cases with the ratio between 10-30 had neovascularization, and the incidence of the latter increased to 82.6% among cases with the ratio over 30. The calibres of 344 retinal arteries and veins in normal subjects and in the affected and unaffected eyes of BRVO patients were measured to find that both vessels were narrowed in late patients of retinal vein occlusion.

Fluorescein Angiography↗