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PubMed1999

Standardisation of DNA quantitation by image analysis: quality control of instrumentation.

BACKGROUND: DNA image analysis is frequently performed in clinical practice as a prognostic tool and to improve diagnosis. The precision of prognosis and diagnosis depends on the accuracy of analysis and particularly on the quality of image analysis systems. It has been reported that image analysis systems used for DNA quantification differ widely in their characteristics (Thunissen et al.: Cytometry 27: 21-25, 1997). This induces inter-laboratory variations when the same sample is analysed in different laboratories. In microscopic image analysis, the principal instrumentation errors arise from the optical and electronic parts of systems. They bring about problems of instability, non-linearity, and shading and glare phenomena. METHODS: The aim of this study is to establish tools and standardised quality control procedures for microscopic image analysis systems. Specific reference standard slides have been developed to control instability, non-linearity, shading and glare phenomena and segmentation efficiency. RESULTS: Some systems have been controlled with these tools and these quality control procedures. Interpretation criteria and accuracy limits of these quality control procedures are proposed according to the conclusions of a European project called PRESS project (Prototype Reference Standard Slide). Beyond these limits, tested image analysis systems are not qualified to realise precise DNA analysis. CONCLUSIONS: The different procedures presented in this work determine if an image analysis system is qualified to deliver sufficiently precise DNA measurements for cancer case analysis. If the controlled systems are beyond the defined limits, some recommendations are given to find a solution to the problem.

DNA, Neoplasm↗

PubMed1994

Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.

A novel automated image analysis method is described for characterizing the viability and morphology of animal cells from suspension cultures. With the aid of the exclusion dye Trypan Blue, the total and viable cell counts and the percentage of dead cells present are found. The area, perimeter, equivalent diameter, and circularity of the projected image of each cell are also measured, allowing the estimation of cell volume. The image analysis method is inherently sensitive, precise, consistent (non-operator dependent) and relatively fast, taking approximately 22 min to analyze one sample. The data it gives on individual cellular activity, as characterized by Trypan Blue uptake and cell morphology, are valuable in allowing early diagnosis of even subtle changes in the health of a culture. The method should permit better optimization of culture conditions, and will provide data for the modeling of cell population dynamics. As the conventional manual method is operator dependent, relatively limited in the amount of information it provides, and has a tendency to underestimate Trypan Blue takeup, it is suggested that image analysis be the preferred option for animal cell counting and viability determinations.

Animals↗

PubMed2002

Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence-based digital image analysis.

Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus. After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen. The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera. Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells. Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis. At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability. The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area). This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor. Estimated viable biomass during fermentation was strongly influenced by the process conditions. The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T. harzianum.

Acridine Orange↗

PubMed1992

Quantitative DNA analysis and proliferation in breast carcinomas. A comparison between image analysis and flow cytometry.

The DNA content and proliferation in 100 invasive breast carcinomas were evaluated by computerized image analysis (IA) and flow cytometry (FCM). For DNA content, image analysis of Feulgen-stained slides of fresh tumor imprints were compared with flow cytometry of propidium iodide-stained disaggregated fresh tumor tissue. The DNA indices obtained by the two methods showed close correlation by linear regression analysis (r = 0.89, p less than .001). There were 44 (44%) diploid and 56 (56%) aneuploid tumors. There was agreement between the two methods in detection of aneuploidy in 81% of tumors. Image analysis required smaller tissue samples, permitted direct visualization and selection of tumor cells, and was more sensitive in detecting tetraploid and highly aneuploid cell populations. In contrast, flow cytometry histograms provided better resolution, and were more effective in detecting multiploid tumors and near-diploid aneuploid tumors. Aneuploidy was significantly related to various adverse prognostic parameters, namely, negative estrogen receptor, high mitotic rate, high histologic and nuclear grades. Proliferation was evaluated by measuring the FCM S phase fraction (SPF), and by image analysis quantitation of immunohistochemical staining using Ki-67 monoclonal antibody. SPF and Ki-67 count showed modest correlation (r = 0.42). Both SPF and Ki-67 count were significantly related to the mitotic rate, histologic and nuclear grades. Our results indicate that the two methods provide comparable results, but offer individual advantages and are complementary techniques in analyzing DNA ploidy and proliferation in breast carcinomas.

Adenocarcinoma, Mucinous↗

PubMed1995

Macroscopic assessment of pulmonary emphysema by image analysis.

AIMS: To propose a computerised image analysis based method for measuring, on paper mounted lung sections, the area macroscopically occupied by emphysema. METHODS: The study was based on the assessment of 69 lung sections prepared following a modified Gough-Wentworth technique. The results obtained from image analysis, point counting, and panel grading methods were compared, as was the repeatability of image analysis and panel grading. RESULTS: The results from image analysis and from point counting were not significantly different (p = 0.609) and significant quadratic regressions (r = 0.96, p < 0.001) were found between measurements from image analysis and from panel grading, the computerised technique being shown to be the most reproducible. CONCLUSIONS: Image analysis is a valuable and reproducible method to measure the area of lung macroscopically involved by emphysema.

Histocytological Preparation Techniques↗

PubMed1990

Quantitative energy-filtered image analysis in cytochemistry. II. Morphometric analysis of element-distribution images.

A combination of energy-filtered electron microscopy (EFEM) and an image-analysis system (IBAS/2000) is used for a morphometric analysis of chemical reaction products in cells. Electron energy-loss spectroscopic element-distribution images are acquired from cytochemical reaction products in a variety of cellular objects: (1) colloidal thorium particles in extra-cellular coat material, (2) iron-containing ferritin particles in liver parenchymal cells, (3) barium-containing reaction products in endoplasmic reticulum stacks, (4) elements present in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. Areas or area fractions are determined from such element-distribution images by application of an objective image segmentation method. By superposition of two or more element-distribution images, mutual element relations are qualitatively established in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. By comparing electron spectroscopic images (ESI) with element-distribution images, the mutual contrast per element relations are quantitatively investigated. The obtained gain in resolution in such electron energy-loss spectroscopic element-distribution images will be explained and discussed.

Animals↗

PubMed1992

Light microscopic morphometric analysis of peroxisomes by automatic image analysis: advantages of immunostaining over the alkaline DAB method.

The feasibility of light microscopic post-embedding immunocytochemistry for morphometry of peroxisomes using automatic image analysis was investigated and compared with the classical alkaline DAB method. Perfusion-fixed rat liver tissue was either embedded in LR White or incubated in the alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase. Sections from the LR White-embedded material were incubated with a monospecific antibody against catalase, followed by protein A-gold and silver intensification. Determination of peroxisomal volume density in sections of different thickness revealed that the values increased with section thickness in DAB-stained sections but were unaffected in immunostained preparations. Moreover, the absolute value for volume density of peroxisomes, as determined by light microscopy in immunostained sections, was quite close to the value obtained by analysis of electron microscopic preparations. Finally, morphometric analysis of bezafibrate-induced peroxisome proliferation revealed that the ratio of proliferation obtained by light microscopy in immunostained sections was very close to the results obtained by electron microscopic morphometry. The main advantage of post-embedding immunostaining for light microscopic morphometry is that it restricts the immunocytochemical reaction product to the surface of the section, thus making it independent of section thickness.

3,3'-Diaminobenzidine↗

PubMed2005

Database design and implementation for quantitative image analysis research.

Quantitative image analysis (QIA) goes beyond subjective visual assessment to provide computer measurements of the image content, typically following image segmentation to identify anatomical regions of interest (ROIs). Commercially available picture archiving and communication systems focus on storage of image data. They are not well suited to efficient storage and mining of new types of quantitative data. In this paper, we present a system that integrates image segmentation, quantitation, and characterization with database and data mining facilities. The paper includes generic process and data models for QIA in medicine and describes their practical use. The data model is based upon the Digital Imaging and Communications in Medicine (DICOM) data hierarchy, which is augmented with tables to store segmentation results (ROIs) and quantitative data from multiple experiments. Data mining for statistical analysis of the quantitative data is described along with example queries. The database is implemented in PostgreSQL on a UNIX server. Database requirements and capabilities are illustrated through two quantitative imaging experiments related to lung cancer screening and assessment of emphysema lung disease. The system can manage the large amounts of quantitative data necessary for research, development, and deployment of computer-aided diagnosis tools.

Algorithms↗

PubMed1992

Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.

Endoplasmic Reticulum↗

PubMed2003

Automated three-dimensional analysis of particle measurements using an optical profilometer and image analysis software.

The automated collection of topographic images from an optical profilometer coupled with existing image analysis software offers the unique ability to quantify three-dimensional particle morphology. Optional software available with most optical profilers permits automated collection of adjacent topographic images of particles dispersed onto a suitable substrate. Particles are recognized in the image as a set of continuous pixels with grey-level values above the grey level assigned to the substrate, whereas particle height or thickness is represented in the numerical differences between these grey levels. These images are loaded into remote image analysis software where macros automate image processing, and then distinguish particles for feature analysis, including standard two-dimensional measurements (e.g. projected area, length, width, aspect ratios) and third-dimensional measurements (e.g. maximum height, mean height). Feature measurements from each calibrated image are automatically added to cumulative databases and exported to a commercial spreadsheet or statistical program for further data processing and presentation. An example is given that demonstrates the superiority of quantitative three-dimensional measurements by optical profilometry and image analysis in comparison with conventional two-dimensional measurements for the characterization of pharmaceutical powders with plate-like particles.

Glass↗

PubMed1996

Breast carcinoma. Correlations between visual diagnostic criteria for histologic grading and features of image analysis.

OBJECTIVE: To investigate the relevance of image analysis for grading breast carcinomas. STUDY DESIGN: The results of histologic grading were correlated with 18 features of image analysis, including SD. "Simple" characteristics, like area and perimeter, shape indices, optical density and textural features of nuclei from cancer cells, were analyzed. Hematoxylin-eosin-stained tissue sections of 67 cancer specimens were routinely used for the study. RESULTS: We found statistically significant correlations between overall histologic grading and the sum of its subscores and features of image analysis, especially nuclear area, nuclear perimeter and the diameter of the circumscribing circle (diametercirc), including their SDs. The visually and therefore subjectively assessed subscore of the nuclear pleomorphism of histologic grading significantly correlated with the features of image analysis, like nuclear area, nuclear perimeter, diametercirc, integrated optical density and correlation (and their SDs). There were significant relationships between the absolute numbers of mitoses per 10 high-power fields and nuclear area, nuclear perimeter and diametercirc (and their SDs). We did not observe a significant correlation between the subscore of tubule formation of histologic grading and any of the features of the image analysis studied. Furthermore, the correlations between the features of image analysis and the subscores of the visual histologic grading system were analyzed with respect to each other. The subscore of nuclear pleomorphism of histologic grading correlated best with overall grading (r = .72), whereas no significant correlation could be found between the subscores of nuclear pleomorphism and mitotic activity. CONCLUSION: Image analysis provides objectivity and reproducibility to the grading of breast carcinomas and thus could contribute to more individualized prognostication of the disease.

Breast Neoplasms↗

PubMed1998

Multivariate image analysis of magnetic resonance images with the direct exponential curve resolution algorithm (DECRA). Part 2: Application to human brain images.

Owing to the heterogeneity of living tissues, it is challenging to quantify tissue properties using magnetic resonance imaging. Within a single voxel, contributions to the signal may result from several types of 1H nuclei with varied chemical (e.g., -CH2-, -OH) and physical environments (e.g., tissue density, compartmentalization). Therefore, mixtures of 1H environments are prevalent. Furthermore, each unique type of 1H environment may possess a unique and characteristic spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). A method for resolving these unique exponentials is introduced in a separate paper (Part 1. Algorithm and Model System) and uses the direct exponential curve resolution algorithm (DECRA). We present results from an analysis of images of the human head comprising brain tissues.

Adult↗

PubMed2000

Grading, image analysis, and stereopsis of digitally compressed fundus images.

PURPOSE: To investigate the effects of image digitization and compression on the ability to identify and quantify features in color fundus photographs. METHODS: Color fundus photographs were digitized as tagged image file format (TIFF) and high-compression (80:1) and low-compression (30:1) joint photographic experts group (JPEG) images. Rerendered images were subjected to standard grading protocols developed for a clinical trial, and digitized images were subjected to image analysis software for drusen identification and quantitation. Re-created stereoscopic images were compared subjectively with originals. RESULTS: Original, TIFF, and low-compression (30:1) JPEG images were virtually indistinguishable when subjected to close scrutiny with magnification. The overall quality of high-compression (80:1) JPEG images and images digitized at 500 dots per inch was markedly reduced. Protocol grading of original and digitized images was highly concordant within the repeatability of multiple grading of original images. The area subtended by drusen differed by less than 1.0% for all uncompressed and compressed image pairs quantified. Stereoscopic information was accurately preserved when compared with originals for TIFF and low-compression JPEG images. CONCLUSIONS: Fundus images can be digitized and stored with significant compression while preserving stereopsis and image quality suitable for quantitative image analysis and semiquantitative grading. Low-compression (30:1) JPEG images may be suitable for archiving and telemedical applications.

Depth Perception↗

PubMed1999

A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis.

Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors and neurotrophin mimetics. The drawbacks to this approach are the difficulties in quantifying the response. Neurite counts are time consuming and labour intensive, and the accuracy is often questionable due to branching and fasciculation of the neurites. We report here a method of semi-quantitative analysis of neurite outgrowth from chick DRG explants, using image analysis to quantify the area occupied by neurites emanating from the ganglion. This method is rapid, takes into account both the length and number of neurites, and is unaffected by neurite fasciculation or branching. Primary explants of chick DRGs were treated with the neurotrophins nerve growth factor (NGF) or neurotrophin-3 (NT-3) and with the compound K252a. K252b was tested for potentiation of the response to NT-3. The results show a dose dependent outgrowth of neurites from explants treated with NGF, NT-3 and K252a, and potentiation of the NT-3 response by K252b. These responses were quantified by neurite area quantification using image analysis. We conclude that neurite area measurement using image analysis provides a robust means of evaluating neurotrophic activity of growth factors and neurotrophin mimetics in vitro.

Animals↗

PubMed1993

Point counting on the Macintosh. A semiautomated image analysis technique.

In image analysis, point counting is used to estimate three-dimensional quantitative parameters from sets of measurements made on two-dimensional images. Point counting is normally conducted either by hand only or manually through a planimeter. We developed a semiautomated, Macintosh-based method of point counting. This technique could be useful for any point counting application in which the image can be digitized. We utilized this technique to demonstrate increased vacuolation in white matter tracts of rat brains, but it could be used on many other types of tissue. Volume fractions of vacuoles within the corpus callosum of rat brains were determined by analyzing images of histologic sections. A stereologic grid was constructed using the Claris MacDraw II software. The grid was modified for optimum line density and size in Adobe Photoshop, electronically superimposed onto the images and sampled using version 1.37 of NIH Image public domain software. This technique was further automated by the creation of a macro (small program) to create the grid, overlay the grid on a predetermined image, threshold the objects of interest and count thresholded objects at intersections of the grid lines. This method is expected to significantly reduce the amount of time required to conduct point counting and to improve the consistency of counts.

Animals↗

PubMed1999

Extending the dynamic range of fibre length and fibre aspect ratios by automated image analysis

An automatic image analysis system has been developed, that is capable of scanning a large sample area (70 mm x 70 mm) using transmitted light microscopy. The analysis, based on transparent algorithms, successfully discriminates between overlapping and touching fibres, so that sample preparation time is minimized. The system utilizes overlapping image frames to reconstruct partial fibre images and impart unique fibre length information in the range micrometres to millimetres. The software has been made adaptable to enable the accurate measurement of fibre lengths for fibres that curve over many millimetres. Data are presented illustrating the accurate measurement of fibre lengths over a large range of fibre aspect ratios.

Journal Article↗

PubMed1992

Computer-generated diagnosis and image analysis. An overview.

Image analysis provides quantitative data on morphology, cytochemical and histochemical reactions, the location of specific events or reaction sites, and statistical descriptions of the spatial distribution of such events relative to histologic structure. Image analytic methodology in correlation with histopathologic knowledge bases is an essential component in the development of an objective basis for histopathologic diagnostic decision making.

Humans↗

PubMed1991

DNA analysis of cardiac myxomas: flow cytometry and image analysis.

Cardiac myxoma is the most common primary tumor of heart, but there is a longstanding controversy over whether it is a true neoplasm or a reactive lesion. We analyzed 24 cardiac myxomas from 22 patients: 22 by DNA flow cytometry and five by image analysis. Two myxomas were aneuploid; one of those analyzed by flow cytometry, and the other by image analysis. Proliferative fractions (S + G2/M) were high in three tumors from patients with multiple myxomas (mean, 15.9%; SD, 4.0%) as compared with 12 solitary uncomplicated myxomas (mean, 7.7%; SD, 6.0%). S-phase and proliferative fractions were low in embolic, recurrent, and solitary myxomas. The presence of aneuploidy in some myxomas supports a neoplastic origin for this tumor.

Adult↗