Comparative analysis of Enterococcus Faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37 C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (mm3), live bacteria biovolume (mm3), and substrate coverage (%) were quantified by using the BioImage_L software. Results obtained were analyzed by nonparametric tests (P = .05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P < .05) and hydroxyapatite the highest at 21 days (P < .05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P < .05). The percentages of coverage were similar among all substrates at 21 days (P > .05), but at 14 days, bovine bone presented the highest coverage (P < .05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated

Análise invasiva e não invasiva do fuso meiótico de oócitos humanos obtidos de ciclos estimulados: dados preliminares

OBJETIVOS: Avaliar a concordância entre as técnicas de microscopia de polarização e microscopia confocal na avaliação do fuso meiótico de oócitos humanos maturados in vivo. MÉTODOS: Estudo prospectivo que avaliou oócitos com o primeiro corpúsculo polar extruído obtidos de mulheres inférteis submetidas à estimulação ovariana para realização de injeção intracitoplasmática de espermatozoide. Os oócitos com o primeiro corpúsculo polar extruído foram avaliados por meio da microscopia de polarização e, imediatamente após, foram fixados e corados para avaliação dos microtúbulos e cromatina pela microscopia confocal de alto desempenho. Foram comparadas as técnicas de microscopia de polarização e confocal, de acordo com a visualização ou não do fuso meiótico pela microscopia de polarização e a presença ou não de anomalias meióticas à análise pela microscopia confocal. Foram calculados os intervalos de confiança, o índice de Kappa e a concordância entre as metodologias, considerando a análise da microscopia de imunofluorescência como padrão-ouro para avaliação de normalidade do fuso e distribuição cromossômica oocitária. RESULTADOS: Observou-se que 72,7% dos oócitos em metáfase II com fuso celular não visível à polarização apresentaram anormalidades meióticas à análise confocal e que 55,6% dos oócitos em metáfase II com fuso celular visível à polarização apresentaram-se como oócitos anormais à análise confocal. Somente 44,4% dos oócitos com fuso celular visível à polarização apresentaram-se como normais à análise confocal. A concordância entre os métodos foi de 51,1% (Kappa: 0,11; IC95% -0,0958 - 0,319). CONCLUSÕES: A baixa concordância entre a microscopia de polarização e a confocal na avaliação do fuso meiótico oocitário sugere que a visualização do fuso meiótico de oócitos humanos em metáfase II pela microscopia de polarização tem limitado o valor preditivo de normalidade meiótica oocitária.

Estudo histológico e comparativo das papilas linguais dos cervídeos Mazama americana e Mazama gouzoubira por microscopia de luz e eletrônica de varredura

O tipo de alimentação depende do ambiente em que o animal se encontra, sendo este um fator responsável pela alteração da morfologia, como a estratificação e o nível de queratinização da língua, e a funcionalidade da mesma. Dentre as estruturas morfológicas funcionais da língua, as papilas vêm merecendo destaque devido a sua estreita relação com a dieta. Foram utilizadas duas espécies de cervídeos: cinco Mazama gouazoubira e duas Mazama americana, dividindo-se a língua em três partes: ápice, corpo e raiz. Analisou comparativamente a língua de duas espécies por meio de microscopia de luz e eletrônica de varredura. As papilas filiforme, fungiforme e valada apresentaram na língua das duas espécies estudadas, e com a mesma distribuição, mudando apenas a quantidade e formato de papilas filiformes no ápice lingual e a quantidade e disposição das papilas valadas na raiz da língua, fator este que pode ser ligado à dieta dos animais. . Além disto, sua distribuição é semelhante à de outras espécies de herbívoros.

Unraveling the Mysterious Role of Palladium in Feigl bis(dimethylglyoximate)nickel(II) Spot Tests by Means of Confocal Raman Microscopy

The add protection effect promoted by traces of PdCl2 in [Ni(dmgH)(2)] spot tests was elucidated from confocal Raman microscopy imaging, which revealed the formation of protecting layers of [Pd(dmgH)(2)] closing the extremities of the [Ni(dmgH)(2)] filaments.

Evaluation of epoxy resin sealer after three root canal filling techniques by confocal laser scanning microscopy

The aim of this study was to evaluate the penetration of endodontic sealer into the dentin tubules, the integrity of the sealer layer perimeter, and the sealer area at the apical third after different filling techniques by confocal laser scanning microscopy (CLSM). Forty-five mandibular premolars were mechanically prepared with ProTaper files, until F5 file. Thereafter, they were filled with an epoxy-resin sealer (AH Plus) mixed with Rhodamine B dye (0.1% proportion) and allocated in three groups: Group 1, single master cone; Group 2, cold lateral compaction; and Group 3, Thermafil. For confocal laser scanning microscopy analysis, the specimens were transversely sectioned at 4 mm from the apex. The images at x10 and x40 were analyzed by Imagetool 3.0 software. Significant differences were not found among the three experimental groups according the dentin-impregnate area by the sealer (P = 0.68) and between the sealer and root canal perimeter (P = 0.18). However, root canal filling techniques were significantly different when apical sealer areas were compared (P = 0.001). Thermafil group showed smaller sealer areas (8.09%) while cold lateral compaction and gutta-percha master cone showed similar areas (17.37 and 21.18%, respectively). The dentin-impregnated area was not dependent on the root canal filling technique. Single master cone, cold lateral condensation and Thermafil techniques presented integrity of the sealer perimeter close to 100% and Thermafil resulted in a significantly thinner sealer layer. Microsc. Res. Tech. 75:12771280, 2012. (C) 2012 Wiley Periodicals, Inc.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

Abstract Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.

Quantification of cellular action fibers alignment from confocal microscopic images.

The cellular rheology has recently undergone a rapid development with particular attention to the cytoskeleton mechanical properties and its main components - actin filaments, intermediate filaments, microtubules and crosslinked proteins. However it is not clear what are the cellular structural changes that directly affect the cell mechanical properties. Thus, in this work, we aimed to quantify the structural rearrangement of these fibers that may emerge in changes in the cell mechanics. We created an image analysis platform to study smooth muscle cells from different arteries: aorta, mammary, renal, carotid and coronary and processed respectively 31, 29, 31, 30 and 35 cell image obtained by confocal microscopy. The platform was developed in Matlab (MathWorks) and it uses the Sobel operator to determine the actin fiber image orientation of the cell, labeled with phalloidin. The Sobel operator is used as a filter capable of calculating the pixel brightness gradient, point to point, in the image. The operator uses vertical and horizontal convolution kernels to calculate the magnitude and the angle of the pixel intensity gradient. The image analysis followed the sequence: (1) opens a given cells image set to be processed; (2) sets a fix threshold to eliminate noise, based on Otsu's method; (3) detect the fiber edges in the image using the Sobel operator; and (4) quantify the actin fiber orientation. Our first result is the probability distribution II(Δθ) to find a given fiber angle deviation (Δθ) from the main cell fiber orientation θ0. The II(Δθ) follows an exponential decay II(Δθ) = Aexp(-αΔθ) regarding to its θ0. We defined and determined a misalignment index α of the fibers of each artery kind: coronary αCo = (1.72 ‘+ or =’ 0.36)rad POT -1; renal αRe = (1.43 + or - 0.64)rad POT -1; aorta αAo = (1.42 + or - 0.43)rad POT -1; mammary αMa = (1.12 + or - 0.50)rad POT -1; and carotid αCa = (1.01 + or - 0.39)rad POT -1. The α of coronary and carotid are statistically different (p < 0.05) among all analyzed cells. We discussed our results correlating the misalignment index data with the experimental cell mechanical properties obtained by using Optical Magnetic Twisting Cytometry with the same group of cells.