The applicability of hematoxylin-eosin staining plus fluorescence or confocal laser scanning microscopy to the study of elastic fibers in cartilages

This study focuses on the use of hemotoxylin-eosin staining plus fluorescence microscopy for the investigation of elastic fibers in some elastic cartilages. We have observed that elastic fibers are consistently imaged by the proposed procedure and the resolution attained is similar to that obtained with the classical Weigert's fuchsin-resorcin. The results also demonstrate that elastin autofluorescence gives little or no contribution to the final fluorescence and that the use of the confocal laser scanning microscope adds to the resolution, permits the use of thicker sections and reveals of minute structural features. We conclude that this is a relevant tool in elastin research.

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin autofluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results.

Indirect georeferencing of terrestrial laser scanning data using control lines

An indirect method for the georeferencing of 3D point clouds obtained with terrestrial laser scanning (TLS) data using control lines is presented. This technique could be used for rapid data acquisition where resources do not permit the use of expensive navigation sensors or the placement of pre-signalised targets. The most important characteristic is the development of a mathematical model based on the principle that the direction vector of the TLS straight line is coplanar with the plane defined by the origin of the TLS system, one endpoint of a control line and the direction vector of the control line in the ground reference coordinate system. The transformation parameters are estimated by minimising the distance between the control lines and their corresponding TLS straight lines. The proposed method was tested using both simulated and real data, and the advantages of this new approach are compared with conventional surveying methods. © 2013 This article is a U.S. Government work and is in the public domain in the USA.

Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy

AimTo evaluate the antibiofilm activity of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) solutions associated with cetrimide (CTR), and QMiX using confocal laser scanning microscopy.MethodologyEnterococcus faecalis (ATCC- 29212) biofilms were induced on bovine dentine blocks for 14days. The dentine blocks containing biofilm were immersed for 1min in the following solutions: 2.5% NaOCl; 2.5% NaOCl+0.2% CTR; 2% CHX; 2% CHX+0.2% CTR; 0.2% CTR; QMiX. After contact with the solutions, the dentine blocks were stained with Live/Dead((R)) BacLight for analysis of the remaining biofilm using confocal laser scanning microscope. Images were evaluated using the BioImage_L software to determine the total biovolume (m(3)), the green biovolume (live cells) (m(3)) and the percentage of substrate coverage (%). The data were subjected to nonparametric statistical test using Kruskal-Wallis and Dunn's tests at 5% significance level.ResultsAfter exposure to irrigants, the total biovolume observed for CHX, CHX+CTR, CTR, QMiX was similar to distilled water (P>0.05). NaOCl and NaOCl+CTR had the lowest total and green biovolume. The CTR and QMiX had intermediate green biovolume, with greater antibacterial activity than CHX and CHX+CTR (P<0.05). The NaOCl and NaOCl+CTR solutions were associated with microorganism removal and substrate cleaning ability.ConclusionsNaOCl and NaOCl+CTR solutions were effective on microorganism viability and were able to eliminate biofilm. The addition of cetrimide did not influence antibacterial activity.

Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive: analysis by SEM and confocal laser scanning microscopy (CLSM)

This study aimed to evaluate the effect of Er:YAG (L) and diamond drills (DD) on: 1) the microshear bond strength (MPa); 2) the adhesive interface of two-step (TS) – Adper Scotchbond Multipurpose and one-step (OS) adhesives – Adper EasyOne, both from 3M ESPE. Material and methods: According to the preparation condition and adhesives, the samples were divided into four groups: DD_TS (control); DD_OS; L_TS and L_OS. 60 bovine incisors were randomly divided into experimental and groups: 40 for microshear bond strength (n = 10) and 20 for the adhesive interface morphology [6 to measure the thickness of the hybrid layer (HL) and length of tags (t) by CLSM (n = 3); 12 to the adhesive interface morphology by SEM (n = 3) and 2 to illustrate the effect of the instruments on dentine by SEM (n = 1)]. To conduct the microshear bond strength test, four cylinders (0.7 mm in diameter and 1 mm in height with area of adhesion of 0.38 mm) were constructed with resin composite (Filtek Z350 XT – 3M ESPE) on each dentin surface treated by either L or DD and after adhesives application. Microshear bond strength was performed in universal testing machine (EMIC 2000) with load cell of 500 kgf and a crosshead speed of 0.5 mm / min. Adhesive interface was characterized by thickness of hybrid layer (HL) and length of tags (t) in nm, with the aid of UTHSCSA ImageTool software. Results: Microshear bond strength values were: L_TS 34.10 ± 19.07, DD_TS 24.26 ± 9.35, L_OS 33.18 ± 12.46, DD_OS 21.24 ± 13.96. Two-way ANOVA resulted in statistically significant differences only for instruments (p = 0.047). Mann-Whitney identified the instruments which determined significant differences for HL thickness and tag length (t). Concerning to the adhesive types, these differences were only observed for (t). Conclusion: It can be concluded that 1) laser Er:YAG results in higher microshear bond strength values regardless of the adhesive system (TS and OS); 2) the tags did not significant affect the microshear bond strength; 3) the adhesive interface was affected by both the instruments for cavity preparation and the type of adhesive system used.

Confocal laser scanning microscopy investigation of bond interfaces involved in fiber glass post cementation

Objective: This confocal microscopy study evaluated the cement/dentin and cement/post interfaces along theroot canalwallswhenfiberglasspostswerebonded to dentin using different types of cements. Material & Methods: Thirty endodontically treated premolars were divided into 3 groups according to the adhesive materials used in the bonding procedure: Prime & Bond 2.1/Self Cure + Enforce, RelyX Unicem and RelyX Luting. Rhodamine B dye was incorporated in the luting materials for the cementation of the fiber glass posts (Exacto, Angelus) to dentin. Three transversal slices (apical, middle and coronal) were examined under confocal laser scanning microscopy. Statistical analysis was performed using the Kappa, Kruskal-Wallis and Dunnet tests, in a significance level of 5%. Results: The Prime & Bond 2.1/Self Cure + Enforce presented a uniform formation of tags in the dentin but gaps in the cement/dentin interface. The RelyX Unicem and RelyX Luting presented an adhesive interface with a fewer amount of gaps, but showed shorter tag formation than the Enforce system. All cements presented the same pattern of bubbles inside the cements. The RelyX Luting presented a greater amount of cracks inside the cement in comparison with the other cements in the coronal third, while no difference was observed between RelyX Unicem and Enforce. The RelyX Luting showed the lowest quantity of cement penetration into the post. Conclusion: In general, the quality of bonding interfaces of fiber posts luted to root canals was affected by both location and type of cement.

Extração automática de contornos de telhados usando dados de varredura a laser e campos randômicos de Markov

This paper proposes a methodology for automatic extraction of building roof contours from a Digital Elevation Model (DEM), which is generated through the regularization of an available laser point cloud. The methodology is based on two steps. First, in order to detect high objects (buildings, trees etc.), the DEM is segmented through a recursive splitting technique and a Bayesian merging technique. The recursive splitting technique uses the quadtree structure for subdividing the DEM into homogeneous regions. In order to minimize the fragmentation, which is commonly observed in the results of the recursive splitting segmentation, a region merging technique based on the Bayesian framework is applied to the previously segmented data. The high object polygons are extracted by using vectorization and polygonization techniques. Second, the building roof contours are identified among all high objects extracted previously. Taking into account some roof properties and some feature measurements (e. g., area, rectangularity, and angles between principal axes of the roofs), an energy function was developed based on the Markov Random Field (MRF) model. The solution of this function is a polygon set corresponding to building roof contours and is found by using a minimization technique, like the Simulated Annealing (SA) algorithm. Experiments carried out with laser scanning DEM's showed that the methodology works properly, as it delivered roof contours with approximately 90% shape accuracy and no false positive was verified.

Uso de dados fotogramétricos no refinamento geométrico de contornos de telhados de edifícios extraídos de dados laser

In this paper, a methodology is proposed for the geometric refinement of laser scanning building roof contours using high-resolution aerial images and Markov Random Field (MRF) models. The proposed methodology takes for granted that the 3D description of each building roof reconstructed from the laser scanning data (i.e., a polyhedron) is topologically correct and that it is only necessary to improve its accuracy. Since roof ridges are accurately extracted from laser scanning data, our main objective is to use high-resolution aerial images to improve the accuracy of roof outlines. In order to meet this goal, the available roof contours are first projected onto the image-space. After that, the projected polygons and the straight lines extracted from the image are used to establish an MRF description, which is based on relations ( relative length, proximity, and orientation) between the two sets of straight lines. The energy function associated with the MRF is minimized by using a modified version of the brute force algorithm, resulting in the grouping of straight lines for each roof object. Finally, each grouping of straight lines is topologically reconstructed based on the topology of the corresponding laser scanning polygon projected onto the image-space. The preliminary results showed that the proposed methodology is promising, since most sides of the refined polygons are geometrically better than corresponding projected laser scanning straight lines.

Confocal Laser Microscopic Analysis of Biofilm on Newer Feldspar Ceramic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Segmentação de dados de perfilamento a laser em áreas urbanas utilizando uma abordagem Bayesiana

In this paper is presented a region-based methodology for Digital Elevation Model segmentation obtained from laser scanning data. The methodology is based on two sequential techniques, i.e., a recursive splitting technique using the quad tree structure followed by a region merging technique using the Markov Random Field model. The recursive splitting technique starts splitting the Digital Elevation Model into homogeneous regions. However, due to slight height differences in the Digital Elevation Model, region fragmentation can be relatively high. In order to minimize the fragmentation, a region merging technique based on the Markov Random Field model is applied to the previously segmented data. The resulting regions are firstly structured by using the so-called Region Adjacency Graph. Each node of the Region Adjacency Graph represents a region of the Digital Elevation Model segmented and two nodes have connectivity between them if corresponding regions share a common boundary. Next it is assumed that the random variable related to each node, follows the Markov Random Field model. This hypothesis allows the derivation of the posteriori probability distribution function whose solution is obtained by the Maximum a Posteriori estimation. Regions presenting high probability of similarity are merged. Experiments carried out with laser scanning data showed that the methodology allows to separate the objects in the Digital Elevation Model with a low amount of fragmentation.

Extração de vias combinando métodos de detecção de regiões e linhas em imagens de intensidade de pulso laser

This paper aims at extracting street centerlines from previously isolated street regions by using the image of laser scanning intensity. In this image, streets are easily identified, since they manifest as dark, elongate ribbons contrasting with background objects. The intensity image is segmented by using the region growing technique, which generates regions representing the streets. From these regions, the street centerlines are extracted in two manners. The first one is through the Steger lines detection method combined with a line length thresholding by which lines being shorter than a minimum length are removed. The other manner is by combining the skeletonization method of regions based on the Medial Axis Transform and with a pruning process to eliminate as much as possible the ramifications. Experiments showed that the Steger-based method provided results better than the method based on skeletonization.

Effect of bleaching on sound enamel and with early artificial caries lesions using confocal laser microscopy

The aim of this study was to evaluate effect of bleaching agents on sound enamel (SE) and enamel with early artificial caries lesions (CL) using confocal laser scanning microscopy (CLSM). Eighty blocks (4 × 5 × 5 mm) of bovine enamel were used and half of them were submitted to a pH cycling model to induce CL. Eight experimental groups were obtained from the treatments and mineralization level of the enamel (SE or CL) (n=10). SE groups: G1 - unbleached (control); G2 - 4% hydrogen peroxide (4 HP); G3 - 4 HP containing 0.05% Ca (Ca); G4 - 7.5% hydrogen peroxide (7.5 HP) containing amorphous calcium phosphate (ACP). CL groups: G5 - unbleached; G6 - 4 HP; G7 - 4 HP containing Ca; G8 - 7.5 HP ACP. G2, G3, G6, G7 were treated with the bleaching agents for 8 h/day during 14 days, while G4 and G8 were exposed to the bleaching agents for 30 min twice a day during 14 days. The enamel blocks were stained with 0.1 mM rhodamine B solution and the demineralization was quantified using fluorescence intensity detected by CLSM. Data were analyzed using ANOVA and Fisher's tests (α=0.05). For the SE groups, the bleaching treatments increased significantly the demineralization area when compared with the unbleached group. In the CL groups, no statistically significant difference was observed (p>0.05). The addition of ACP or Ca in the composition of the whitening products did not overcome the effects caused by bleaching treatments on SE and neither was able to promote remineralization of CL.

Photogrammetric refinement of laser-based building roof contours

In this paper a photogrammetric method is proposed for refining 3D building roof contours extracted from airborne laser scanning data. It is assumed that laser-derived planar faces of roofs are potentially accurate, while laser-derived building roof contours are not well defined. First, polygons representing building roof contours are extracted from a high-resolution aerial image. In the sequence, straight-line segments delimitating each building roof polygon are projected onto the corresponding laser-derived roof planes by using a new line-based photogrammetric model. Finally, refined 3D building roof contours are reconstructed by connecting every pair of photogrammetrically- projected adjacent straight lines. The obtained results showed that the proposed approach worked properly, meaning that the integration of image data and laser scanning data allows better results to be obtained, when compared to the results generated by using only laser scanning data. © 2013 IEEE.

Detecção de regiões de via integrando dados de varredura a LASER aerotransportado e imagens de alta resolução

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Reconstrução de contornos e cumeeiras de telhados de edifícios a partir de imagens aéreas de alta-resolução e poliedros representativos de edifícios extraídos de dados LASER

Pós-graduação em Ciências Cartográficas - FCT