A statistical modeling approach to computer-aided quantification of dental biofilm.

Biofilm is a formation of microbial material on tooth substrata. Several methods to quantify dental biofilm coverage have recently been reported in the literature, but at best they provide a semiautomated approach to quantification with significant input from a human grader that comes with the grader's bias of what is foreground, background, biofilm, and tooth. Additionally,human assessment indices limit the resolution of the quantification scale; most commercial scales use five levels of quantification for biofilm coverage (0%, 25%, 50%, 75%, and 100%). On the other hand, current state-of-the-art techniques in automatic plaque quantification fail to make their way into practical applications owing to their inability to incorporate human input to handle misclassifications. This paper proposes a new interactive method for biofilm quantification in Quantitative light-induced fluorescence(QLF) images of canine teeth that is independent of the perceptual bias of the grader. The method partitions a QLF image into segments of uniform texture and intensity called superpixels; every superpixel is statistically modeled as a realization of a single 2-D Gaussian Markov random field (GMRF) whose parameters are estimated; the superpixel is then assigned to one of three classes (background, biofilm, tooth substratum) based on the training set of data. The quantification results show a high degree of consistency and precision. At the same time, the proposed method gives pathologists full control to postprocess the automatic quantification by flipping misclassified superpixels to a different state (background,tooth, biofilm) with a single click, providing greater usability than simply marking the boundaries of biofilm and tooth as done by current state-of-the-art methods.

BiofilmQuant: a computer-assisted tool for dental biofilm quantification.

Dental biofilm is the deposition of microbial material over a tooth substratum. Several methods have recently been reported in the literature for biofilm quantification; however, at best they provide a barely automated solution requiring significant input needed from the human expert. On the contrary, state-of-the-art automatic biofilm methods fail to make their way into clinical practice because of the lack of effective mechanism to incorporate human input to handle praxis or misclassified regions. Manual delineation, the current gold standard, is time consuming and subject to expert bias. In this paper, we introduce a new semi-automated software tool, BiofilmQuant, for dental biofilm quantification in quantitative light-induced fluorescence (QLF) images. The software uses a robust statistical modeling approach to automatically segment the QLF image into three classes (background, biofilm, and tooth substratum) based on the training data. This initial segmentation has shown a high degree of consistency and precision on more than 200 test QLF dental scans. Further, the proposed software provides the clinicians full control to fix any misclassified areas using a single click. In addition, BiofilmQuant also provides a complete solution for the longitudinal quantitative analysis of biofilm of the full set of teeth, providing greater ease of usability.

Application of detector precision characteristics and histogram packing for compression of biological fluorescence micrographs.

Modern applications of biological microscopy such as high-content screening (HCS), 4D imaging, and multispectral imaging may involve collection of thousands of images in every experiment making efficient image-compression techniques necessary. Reversible compression algorithms, when used with biological micrographs, provide only a moderate compression ratio, while irreversible techniques obtain better ratios at the cost of removing some information from images and introducing artifacts. We construct a model of noise, which is a function of signal in the imaging system. In the next step insignificant intensity levels are discarded using intensity binning. The resultant images, characterized by sparse intensity histograms, are coded reversibly. We evaluate compression efficiency of combined reversible coding and intensity depth-reduction using single-channel 12-bit light micrographs of several subcellular structures. We apply local and global measures of intensity distribution to estimate maximum distortions introduced by the proposed algorithm. We demonstrate that the algorithm provides efficient compression and does not introduce significant changes to biological micrographs. The algorithm preserves information content of these images and therefore offers better fidelity than standard irreversible compression method JPEG2000.

The expression of connexin 43 in children with Tetralogy of Fallot.

Abnormalities in the expression and distribution of Connexin 43 (Cx43) in cardiomyocytes may lead to anomalous conotruncal embryogenesis and disturbances in the maturation and function of the heart. Tetralogy of Fallot (TOF) is the most frequent, cyanotic congenital heart defect in which conotruncal anomalies, right ventricle dysfunction and life-threatening arrhythmias occur. In this study, age-related changes in the expression and spatial distribution of Cx43 in cardiomyocytes from TOF children compared to patients without right ventricular outflow tract pathology were determined Confocal microscopy and flow cytometry were used to assess the changes. Disturbances in both the expression and distribution of Cx43 were found. In the group of infants with TOF, a lower level of expression of the protein was determined. Cardiomyocytes from TOF hearts were found to have Cx43 distributed over their entire surface, which is the pattern seen in immature tissue. In the controls, Cx43 was located within the intercalated disks. Expression of Cx43 in TOF hearts increases with the age of the subject, whereas its spatial distribution remains the same in both infants and older children. Disturbances in Cx43 expression and localization may influence heart embryogenesis and maturation, contribute to hypertrophy and dysfunction of the right ventricle and induce arrhythmias in children with TOF. Early redistribution of Cx43 and functional maturation of the heart muscle support a strategy of early total correction of the defect.

Spreading-independent growth of normal fibroblasts in three-dimensional cultures.

Changes of cell shape resulting from cellular flattening on culture substratum have previously been demonstrated to correlate with mitotic activity of normal animal cells in monolayer cultures. Here, we compared the shapes and proliferation of chick embryo fibroblasts cultured either in multicellular, multilayered sheets extended between glass fibres, or in standard monolayers. Fibroblasts in sheets retained the mitotic activity characteristic of that observed in sparse monolayer cultures, i.e. considerably higher that in confluent monolayers. Morphometric analyses revealed, however, that the cells in sheets were considerably less flattened than in monolayer cultures. These observations indicate that the modulation of culture conditions resulting in multidirectional cell stretching leads to the dissociation of flattening and mitotic activity of normal animal cells, so long as an intracellular stress field, generated by contractile cytoskeleton and stabilised by intercellular contacts, is maintained.

Comparison of daunomycin effects on human keratinocytes and melanoma HTB 1410 cells. Image cytometry study.

Interacting melanocytes and keratinocytes in the epidermis create a unique structure in which keratinocytes regulate the growth of melanocytes and the expression of cell surface molecules. The critical role of communication between these cells means that any anti-melanoma drug should be studied in the context of its possible influence on keratinocytes. For that reason, this study focused on comparing the influence of daunomycin on human melanoma cells and on keratinocytes in vitro. The effects were studied by cytochemical methods (TUNEL, FITC-Annexin V labelling, endocytosis activity assay, measurements of DNA content) and morphological methods (measurements of cell surface area, perimeter, extension, dispersion and elongation) to verify the hypothesis of differential response. The results of our research demonstrate that keratinocytes are less susceptible than melanoma cells to daunomycin treatment in vitro. Keratinocytes are also able to resume growth when the drug is removed from the medium, whereas melanoma cells have not demonstrated this capacity. Apoptosis was identified as the mechanism by which the drug exerts its cytostatic effects.

Analysis of orientations of collagen fibers by novel fiber-tracking software.

Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to not only its composition but also its structure. This article integrates confocal microscopy imaging and image-processing techniques to analyze the microstructural properties of ECM. This report describes a two- and three-dimensional fiber middle-line tracing algorithm that may be used to quantify collagen fibril organization. We utilized computer simulation and statistical analysis to validate the developed algorithm. These algorithms were applied to confocal images of collagen gels made with reconstituted bovine collagen type I, to demonstrate the computation of orientations of individual fibers.

Three-dimensional visualization of connexin 43 on the human cardiomyocytes.

Gap junctions created by a family of connexin proteins play an important role in the development of human heart. It has been previously shown that the abnormalities of right ventricular outflow tract are related to an altered level of expression of connexin 43. The right ventricular outflow tract narrowing, stenosis, or atresia of the main pulmonary artery and hypertrophy of the right ventricle are observed in tetralogy of Fallot. The aim of the current study was to determine the distribution of connexin 43 on the surface of human cardiomyocytes obtained during reparative surgery for tetralogy of Fallot. Connexin 43 distribution in these cells was compared with distribution of connexin 43 in cardiomyocytes obtained from patients without right ventricular outflow tract pathology. Cardiomyocytes isolated from tissue biopsy were cultured on collagen substratum, fixed with paraformaldehyde, and incubated with goat antihuman connexin 43 antibodies and secondary donkey antigoat antibodies conjugated with fluorescent indocarbocyanine. Z-series of optical sections were recorded using a laser scanning confocal microscope. Three-dimensional data stacks were visualized using volume-rendering techniques. Images of connexin 43 fluorescence revealed a pattern of three-dimensional distribution of connexin on the surface of an individual cardiomyocyte. Cardiomyocytes from tetralogy of Fallot and hearts with normal right ventricular outflow tract differ in the organization of connexin 43. Cardiomyocytes from tetralogy of Fallot hearts revealed disturbed distribution of connexin 43. The protein is located irregularly on the entire surface of the cell. In the controls, connexin 43 can be visualized within the intercalated disks only. These disturbances may influence heart maturation, cause hypertrophy of the right ventricle, and induce severe arrhythmias in children with tetralogy of Fallot.

Interaction of maize zein with wheat gluten in composite dough and bread as determined by confocal laser scanning microscopy.

Protein body-free maize zein, when mixed at 35 degrees C (above its glass transition temperature range), significantly (p < 0.01) improved the rheological and leavening properties of sorghum-wheat composite flour dough, resulting in improved loaf volume. Confocal laser scanning microscopy was used to observe the structure of zein fibrils and the interaction between zein and gluten proteins in the composite dough and bread systems. Autofluorescence and immunolocalization techniques were used to locate gluten and zein, respectively. Optical sections were collected every 0.4 microm through the samples and digitally processed to produce reconstructed three-dimensional images. Results showed that zein fibrils form an outer layer that intermittently coats the gluten networks, thereby strengthening them. This type of microstructure is able to withstand the pressure exerted by gas cell expansion during yeast fermentation to increase loaf volume.