Evaluation of follicular lymphoid depletion in the Bursa of Fabricius: an alternative methodology using digital image analysis and artificial neural networks

Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89% and 96%, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90% and obtained the maximum value for the specificity of 92%. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

Microscopy image segmentation by active contour models

In this thesis a semi-automated cell analysis system is described through image processing. To achieve this, an image processing algorithm was studied in order to segment cells in a semi-automatic way. The main goal of this analysis is to increase the performance of cell image segmentation process, without affecting the results in a significant way. Even though, a totally manual system has the ability of producing the best results, it has the disadvantage of taking too long and being repetitive, when a large number of images need to be processed. An active contour algorithm was tested in a sequence of images taken by a microscope. This algorithm, more commonly known as snakes, allowed the user to define an initial region in which the cell was incorporated. Then, the algorithm would run several times, making the initial region contours to converge to the cell boundaries. With the final contour, it was possible to extract region properties and produce statistical data. This data allowed to say that this algorithm produces similar results to a purely manual system but at a faster rate. On the other hand, it is slower than a purely automatic way but it allows the user to adjust the contour, making it more versatile and tolerant to image variations.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

Image Analysis for the Characterization of Materials for Highway Construction : Phase I, Progress Report, HR-346, 1992

The major objective of this project is to evaluate image analysis for characterizing air voids in Portland cement contract (PCC) and asphalt concrete (AC) and aggregate gradation in asphalt concrete. Phase 1 of this project has concentrated on evaluation and refinement of sample preparation techniques, evaluation of methods and instruments for conducting image analysis, and finally, analysis and comparison of a select portion of samples. Preliminary results suggest a strong correlation between the results obtained from the linear traverse method and image analysis methods for determining percent air voids in concrete. Preliminary work with asphalt samples has shown that damage caused by a high vacuum of the conventional scanning electron microscope (SEM) may too disruptive. Alternative solutions have been explored, including confocal microscopy and low vacuum electron microscopy. Additionally, a conventional high vacuum SEM operating at a marginal operating vacuum may suffice.

Particle shape and orientation in laser diffraction and static image analysis size distribution analysis of micrometer sized rectangular particles

Laser diffraction (LD) and static image analysis (SIA) of rectangular particles [United States Pharmacopeia, USP30-NF25, General Chapter <776>, Optical Miroscopy.] have been systematically studied. To rule out sample dispersion and particle orientation as the root cause of differences in size distribution profiles, we immobilize powder samples on a glass plate by means of a dry disperser. For a defined region of the glass plate, we measure the diffraction pattern as induced by the dispersed particles, and the 2D dimensions of the individual particles using LD and optical microscopy, respectively. We demonstrate a correlation between LD and SIA, with the scattering intensity of the individual particles as the dominant factor. In theory, the scattering intensity is related to the square of the projected area of both spherical and rectangular particles. In traditional LD the size distribution profile is dominated by the maximum projected area of the particles (A). The diffraction diameters of a rectangular particle with length L and breadth B as measured by the LD instrument approximately correspond to spheres of diameter ØL and ØB respectively. Differences in the scattering intensity between spherical and rectangular particles suggest that the contribution made to the overall LD volume probability distribution by each rectangular particle is proportional to A2/L and A2/B. Accordingly, for rectangular particles the scattering intensity weighted diffraction diameter (SIWDD) explains an overestimation of their shortest dimension and an underestimation of their longest dimension. This study analyzes various samples of particles whose length ranges from approximately 10 to 1000 μm. The correlation we demonstrate between LD and SIA can be used to improve validation of LD methods based on SIA data for a variety of pharmaceutical powders all with a different rectangular particle size and shape.

Evaluation of follicular lymphoid depletion in the Bursa of Fabricius: an alternative methodology using digital image analysis and artificial neural networks

Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89% and 96%, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90% and obtained the maximum value for the specificity of 92%. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

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

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

Confocal and two-photon microscopy : image enhancement /

Confocal and two-photon microcopy have become essential tools in biological research and today many investigations are not possible without their help. The valuable advantage that these two techniques offer is the ability of optical sectioning. Optical sectioning makes it possible to obtain 3D visuahzation of the structiu-es, and hence, valuable information of the structural relationships, the geometrical, and the morphological aspects of the specimen. The achievable lateral and axial resolutions by confocal and two-photon microscopy, similar to other optical imaging systems, are both defined by the diffraction theorem. Any aberration and imperfection present during the imaging results in broadening of the calculated theoretical resolution, blurring, geometrical distortions in the acquired images that interfere with the analysis of the structures, and lower the collected fluorescence from the specimen. The aberrations may have different causes and they can be classified by their sources such as specimen-induced aberrations, optics-induced aberrations, illumination aberrations, and misalignment aberrations. This thesis presents an investigation and study of image enhancement. The goal of this thesis was approached in two different directions. Initially, we investigated the sources of the imperfections. We propose methods to eliminate or minimize aberrations introduced during the image acquisition by optimizing the acquisition conditions. The impact on the resolution as a result of using a coverslip the thickness of which is mismatched with the one that the objective lens is designed for was shown and a novel technique was introduced in order to define the proper value on the correction collar of the lens. The amoimt of spherical aberration with regard to t he numerical aperture of the objective lens was investigated and it was shown that, based on the purpose of our imaging tasks, different numerical apertures must be used. The deformed beam cross section of the single-photon excitation source was corrected and the enhancement of the resolution and image quaUty was shown. Furthermore, the dependency of the scattered light on the excitation wavelength was shown empirically. In the second part, we continued the study of the image enhancement process by deconvolution techniques. Although deconvolution algorithms are used widely to improve the quality of the images, how well a deconvolution algorithm responds highly depends on the point spread function (PSF) of the imaging system applied to the algorithm and the level of its accuracy. We investigated approaches that can be done in order to obtain more precise PSF. Novel methods to improve the pattern of the PSF and reduce the noise are proposed. Furthermore, multiple soiu'ces to extract the PSFs of the imaging system are introduced and the empirical deconvolution results by using each of these PSFs are compared together. The results confirm that a greater improvement attained by applying the in situ PSF during the deconvolution process.

Clustering algorithms and shape factor methods to discriminate among small GTPase phenotypes using DIC image analysis.

Naïvement perçu, le processus d’évolution est une succession d’événements de duplication et de mutations graduelles dans le génome qui mènent à des changements dans les fonctions et les interactions du protéome. La famille des hydrolases de guanosine triphosphate (GTPases) similaire à Ras constitue un bon modèle de travail afin de comprendre ce phénomène fondamental, car cette famille de protéines contient un nombre limité d’éléments qui diffèrent en fonctionnalité et en interactions. Globalement, nous désirons comprendre comment les mutations singulières au niveau des GTPases affectent la morphologie des cellules ainsi que leur degré d’impact sur les populations asynchrones. Mon travail de maîtrise vise à classifier de manière significative différents phénotypes de la levure Saccaromyces cerevisiae via l’analyse de plusieurs critères morphologiques de souches exprimant des GTPases mutées et natives. Notre approche à base de microscopie et d’analyses bioinformatique des images DIC (microscopie d’interférence différentielle de contraste) permet de distinguer les phénotypes propres aux cellules natives et aux mutants. L’emploi de cette méthode a permis une détection automatisée et une caractérisation des phénotypes mutants associés à la sur-expression de GTPases constitutivement actives. Les mutants de GTPases constitutivement actifs Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L et Rsr1 G12V ont été analysés avec succès. En effet, l’implémentation de différents algorithmes de partitionnement, permet d’analyser des données qui combinent les mesures morphologiques de population native et mutantes. Nos résultats démontrent que l’algorithme Fuzzy C-Means performe un partitionnement efficace des cellules natives ou mutantes, où les différents types de cellules sont classifiés en fonction de plusieurs facteurs de formes cellulaires obtenus à partir des images DIC. Cette analyse démontre que les mutations Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L et Rsr1 G12V induisent respectivement des phénotypes amorphe, allongé, rond et large qui sont représentés par des vecteurs de facteurs de forme distincts. Ces distinctions sont observées avec différentes proportions (morphologie mutante / morphologie native) dans les populations de mutants. Le développement de nouvelles méthodes automatisées d’analyse morphologique des cellules natives et mutantes s’avère extrêmement utile pour l’étude de la famille des GTPases ainsi que des résidus spécifiques qui dictent leurs fonctions et réseau d’interaction. Nous pouvons maintenant envisager de produire des mutants de GTPases qui inversent leur fonction en ciblant des résidus divergents. La substitution fonctionnelle est ensuite détectée au niveau morphologique grâce à notre nouvelle stratégie quantitative. Ce type d’analyse peut également être transposé à d’autres familles de protéines et contribuer de manière significative au domaine de la biologie évolutive.

A novel method for measuring lag times in division of individual bacterial cells using image analysis

A method is presented for determining the time to first division of individual bacterial cells growing on agar media. Bacteria were inoculated onto agar-coated slides and viewed by phase-contrast microscopy. Digital images of the growing bacteria were captured at intervals and the time to first division estimated by calculating the "box area ratio". This is the area of the smallest rectangle that can be drawn around an object, divided by the area of the object itself. The box area ratios of cells were found to increase suddenly during growth at a time that correlated with cell division as estimated by visual inspection of the digital images. This was caused by a change in the orientation of the two daughter cells that occurred when sufficient flexibility arose at their point of attachment. This method was used successfully to generate lag time distributions for populations of Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa, but did not work with the coccoid organism Staphylococcus aureus. This method provides an objective measure of the time to first cell division, whilst automation of the data processing allows a large number of cells to be examined per experiment. (c) 2005 Elsevier B.V. All rights reserved.

Image analysis of modified cellulose fibers from sugarcane bagasse by zirconium oxychloride

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A method for the digital image analysis of ceramic grains based on shape factor segmentation

A very simple and robust method for ceramics grains quantitative image analysis is presented. Based on the use of optimal imaging conditions for reflective light microscopy of bulk samples, a digital image processing routine was developed for shading correction, noise suppressing and contours enhancement. Image analysis was done for grains selected according to their concavities, evaluated by perimeter ratio shape factor, to avoid consider the effects of breakouts and ghost boundaries due to ceramographic preparation limitations. As an example, the method was applied for two ceramics, to compare grain size and morphology distributions. In this case, most of artefacts introduced by ceramographic preparation could be discarded due to the use of perimeter ratio exclusion range.

A New Technique for Firn Grain-Size Measurement Using SEM Image Analysis

Firn microstructure is accurately characterized using images obtained from scanning electron microscopy (SEM). Visibly etched grain boundaries within images are used to create a skeleton outline of the microstructure. A pixel-counting utility is applied to the outline to determine grain area. Firn grain sizes calculated using the technique described here are compared to those calculated using the techniques of Cow (1969) and Gay and Weiss (1999) on samples of the same material, and are found to be substantially smaller. The differences in grain size between the techniques are attributed to sampling deficiencies (e.g. the inclusion of pore filler in the grain area) in earlier methods. The new technique offers the advantages of greater accuracy and the ability to determine individual components of the microstructure (grain and pore), which have important applications in ice-core analyses. The new method is validated by calculating activation energies of grain boundary diffusion using predicted values based on the ratio of grain-size measurements between the new and existing techniques. The resulting activation energy falls within the range of values previously reported for firn/ice.

3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms.