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Deformable models as a tool for biometric and histopathological applications
- M.A.G. López, N.G. Posada, J.R. Gadelha, F. Morgado
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 75-76
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- August 2013
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Mathematical morphology is a novel geometry-based technique for image processing and analysis, originally developed to process binary images, based on the use of simple concepts from set theory and geometry such as set inclusion, intersection, union, complementation, and translation Geometry is used to represent object shape, physics inflict constraints on how the shape may vary over space and time, and optimal approximation theory make available the formal underpinnings of mechanisms for fitting the models to measured data. Deformable curve, surface, and solid models gained popularity after they were proposed by Terzopoulos for use in computer vision and computer graphics in the mid 1980’s introducing the theory of continuous (multidimensional) deformable models in a Lagrangian dynamics setting, based on deformation energies in the form of generalized splines. The deformable model that has attracted the most attention to date is popularly known as “snakes”, planar deformable contours that are useful in several image analysis tasks. They are often used to approximate the locations and shapes of object boundaries in images based on the reasonable assumption that boundaries are piecewise continuous or smooth.
This work presents a novel approach since it joins (and, indeed, reinforces) the index framework with the evaluation of the same biological samples by a suitable combination of deformable models. Nucleus contour is identified through Active Shape Models techniques, and cytoplasm contour’s detected through parametric Snakes, with prior image preprocessing based on statistical and mathematical morphology techniques. Morphometric parameters such as nucleus and cytoplasm area and ratio between them are then easily computed. Biometry was performed using an ocular micrometer and nucleus/cytoplasm ratios were obtained characterizing each of the three identified stages: Immature, Vitellogenic and Mature. This resulted in a collection of tools, called morphological operators, which are eminently suited for the analysis of shape and structure in binary images. Acartia tonsa was used as model to establish an index for oocyte maturity determination based in citometry and histochemical evaluation of gonadic masses. It was validated the application of a methodology with a realistic background and a new, more accurate and ecologically realistic index for oocyte staging emerged.
Histology and histochemistry of the reproductive potential of Acartia clausi (copepoda: calanoida)
- F. Morgado, S. Terdalkar, J.R. Gadelha, M.L. Pereira
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 91-92
- Print publication:
- August 2013
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The seasonal fluctuations in zooplankton densities in temperate climates have been long known and the multiplicity of performed studies identified a vast number of factors responsible for these phenomena, such as changes in the physico-chemical factors and other such types of environmental forces governing them. Acartia clausi is a euryaline temperate-boreal species very common in the Portuguese coastal ecosystems, in both estuarine and coastal waters. It is usually described as a temperate water species of neritic calanoid copepod, which is associated with warmer water regions, and as a result becomes more abundant in the summer months, reaching a biomass maximum during the months of July and August. Growth and egg production have been studied extensively in some Acartia species. In the present study histology and histochemistry were selected to determine the reproductive potential of A. clausi.
The ovigerous females were identified with a binocular microscope, isolated and fixed in the Bouin’s solution for histological (5 om thickness, mounting and Haematoxylin - Eosin staining) and histochemical analysis (Periodic Acid Schiff method (PAS) with Haematoxylin as a counter stain for the identification of the carbohydrate content and vitellogenic oocytes. The size of the oocytes was evaluated through measurements made with a micrometer.
The microscopic studies and Image analysis indicated that, in the month of September, the majority of the oocytes were immature and had reduced or almost negligible carbohydrate contents with very few vitellogenic oocytes (Figure 1 A and B), while the specimens from the month of March exhibited a large difference in the oocyte dimensions. These were mature and more vitellogenic and occupied almost half the volume of the body (Figure 1 C, D and E). This shows that, during the month of September, the environmental conditions are not favorable for the maturation of gonads in these species while in the month of March they proliferate and the species shows a high degree of reproductive potential.
This work forms a valid approach in understanding the population fluctuations and reproductive status in a key species of copepod showing a particular temporal variation associated with its reproductive strategy.
A novel formed classifier for analyzing cellular contents in cells images
- M.A.G. López, N.G. Posada, J.R. Gadelha, F. Morgado
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 65-66
- Print publication:
- August 2013
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Several approaches for cell image segmentation have been proposed and applied with success in the biomedical sciences field, but cell content estimation is still a not solved problem. This paper presents a novel formed classifier that combine appropriately “intelligent scissors” (livewire), object’s pixel information (RGB values of colors) and probabilistic artificial neural networks (ANN) techniques for segmenting and estimating precisely the cytoplasm composition in relation to protein, lipid, and carbohydrates. Based on this it is possible to estimate the gonad’s state of development and the maturation stage of oocytes in adult ovigerous females gonads.
The ovigerous mature females (identifiable by their brood pouch – empty, with eggs, with eyeless or eyed embryos) were selected under a dissecting microscope in the laboratory. A total of 170 gonadic masses from females containing oocytes in different developmental stages were analyzed. A total of 100 sections were observed by light microscopy in order to record the arrangement of the ovaries and determine the maturity stage of the various germ cells.
With the ANN network (Fig. 1) trained is possible to carry out the classification step. Classification aims to associate each cytoplasm pixel to one of the selected classes. In this process each cytoplasm pixel is simulated (Figure 2 A). As result a new image containing the classified cytoplasm is produced, where each class is represented by a different colour level (Figure 2 B). Figure 2 C show from left to right the full “classification flow process” including ROI selection, cytoplasm and nucleus contour extraction, cytoplasm segmentation and cytoplasm content classification. The proposed classifier was tested successfully in a representative image dataset, demonstrating the effectiveness and robustness of this new method facilitating the histochemical analysis of cell contents, based on a gonads model.
Histology and histochemistry of sea anemones in environmental contamination studies
- J.R. Gadelha, F. Morgado, A.M.V.M. Soares
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 57-58
- Print publication:
- August 2013
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Since contaminants such as metals, POPs (Persistent Organic Pollutants) and PAHs (Polycyclic aromatic compounds represent a risk to human health and to the environment, it is therefore extremely necessary to study their biological effects. Man-made chemicals endocrine disruptors such as estrogens pose the potential to modulate endocrine function and thus adversely affect humans and other animal’s reproductive development. In this work, sublethal toxicity tests were carried out with the sea anemones Actinia equina and Anemonia sulcata exposed to 17-β-Estradiol. A. equina and A. sulcata are species that present a wide geographic distribution and might possibly be effective pollution indicators. Histological and histochemical techniques were used to detect morphological changes in sea anemones in order to find histological parameters that could be useful as early biomarkers of environmental contamination. The histological and histochemical procedures followed by standard methods to Hematoxylin and eosine (H & E), Periodic acid Shiff reagent (PAS) and Masson`s Tricrome (TMass) stained adaptated to Actiniidae conditions. Such as, the fixation (formalin and alcohol) time is high (96 hours), because this organisms are almost 98% of water body constitution. The slides obtained were observed by light microscopy means. The assemblage of methodologies described permitted the identification of several anomalies/pathologies in different parts of the sea anemones body, with special attention to reproductive structures. Results obtained for A. sulcata showed vitellogenic oocytes with anomalous dimensions, altered cytoplasm or without cellular membrane limits (Figure 1). It can also be observed lipid accumulations and cells membranes not always preserved. In certain areas oocytes presents small reactivity with atypical PAS low basophilic patterns. In the mesoglea the amoebocytes showed more eosinophilic cytoplasm or extracellular bodies suggesting necrosis or protein content. The effects at 10 µg/L concentrations show a considerable number of oocytes with germinal vesicles membranes and indistinct cytoplasm boundaries (Figure 1). Results obtained for A. equina showed some morphological changes in the spermatocytes of male gonads and in the germinal vesicles the female gonads. The effects observed at higher concentrations shows oocytes and ovarian tissues disintegration. The morphologic alterations observed suggested a delay in spermatogenesis and although there have been no alterations in female vitellogenic granules, there are changes in their maturation. The whole effects lead to verify the role that the estradiol in the Anthozoan reproductive system.
Acknowledgments: This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.
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