Optical Measurement of the Stoichiometry of Thin-Film Compounds Synthetized From Multilayers: Example of Cu(In,Ga)Se2.

The properties of centimeter-sized thin-film compound semiconductors depend upon the morphology and chemical composition of the multiple submicrometer-thick elemental and alloy precursor layers from which they are synthesized. The challenge is to characterize the individual precursor layers over these length scales during a multistep synthesis without altering or contaminating them. Conventional electron and X-ray-based morphological and compositional techniques are invasive, require preparation, and are thus incompatible with in-line synthesis processes. In a proof-of-concept study, we applied confocal laser scanning microscopy (CLSM) as a noninvasive optical imaging technique, which measures three-dimensional surface profiles with nanoscale resolution, to this challenge. Using an array of microdots containing Cu(In,Ga)Se2 semiconductor layers for solar cells as an example, we performed CLSM correlative studies to quantify morphological and layer thickness changes during four stages of a thin-film compound synthesis. Using simple assumptions, we measured the micrometer-scale spatially resolved chemical composition of stacked precursor layers to predict the final material phases formed and predict relative device performance. The high spatial resolution, coupled with the ability to measure sizeable areas without influencing the synthesis at high speed, makes CLSM an excellent prospect for research and quality control tool for thin films.

Effects of DJK-5 and chlorhexidine on exopolysaccharide volume and pH in oral biofilms.

BACKGROUND: Exopolysaccharides (EPS) are essential constituents of the extracellular matrix within oral biofilms and are significantly influenced by the local microenvironment. This study aimed to investigate the impact of two distinct antimicrobial agents, DJK-5 and chlorhexidine (CHX), on the EPS volume and pH levels in oral biofilms. METHODS: Oral biofilms obtained from two donors were cultured on hydroxyapatite discs for durations of 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks. Subsequently, these biofilms were subjected to treatment with 10 µg/mL DJK-5 or 2% CHX for 3 min. The impact of these antimicrobial treatments on factors such as the proportion of dead bacterial, in situ pH, and EPS volume within the biofilms was assessed using corresponding fluorescent probes. The examination was carried out utilizing confocal laser scanning microscopy, and the resulting images were analyzed with a focus on the upper and lower layers of the biofilm, respectively. RESULTS: DJK-5 exhibited a more potent bactericidal effect compared to CHX across the 3-day to 4-week duration of the biofilm (P < 0.05). The biofilms were acidic, with the upper layer being less acidic than the lower layer (P < 0.05). Both antimicrobial agents increased the pH, but DJK-5 had a greater effect than CHX (P < 0.05). The volume of EPS was significantly lower in DJK-5 treated biofilms compared to that of CHX, regardless of age or layer (P < 0.05). CONCLUSION: DJK-5 exhibited superior effectiveness in reducing viable bacteria and EPS volume, as well as in raising extracellular pH, as compared to chlorhexidine.

In vitro evaluation of the antibacterial effect of four root canal sealers on dental biofilms.

OBJECTIVES: To dynamically evaluate the effect of four root canal sealers on the killing of biofilms within dentinal tubules. MATERIALS AND METHODS: Dentin blocks were prepared for infection of the dentinal tubules. Enterococcus faecalis VP3-181 and multi-species bacteria from two donors were cultured. After 3 days of incubation, the infected dentin specimens were rinsed with sterile water for 1 min and subjected to treatment. Additionally, multi-species bacteria from donor 1 were incubated for 3 weeks to allow biofilm maturation and then the specimens were subjected to treatment. Gutta-percha-treated dentin specimens comprised the control group. A root canal sealer (bioceramic sealers: EndoSequence BC Sealer, ProRoot Endo Sealer, or GuttaFlow Bioseal; and a traditional silicone-based sealer: Guttaflow 2) was spread onto the canal walls of the dentin. The specimens were examined with confocal laser scanning microscopy at 7, 30, or 60 days. RESULTS: In the 3-day-old biofilm group, the proportion of killed bacteria decreased significantly from the first 7 days of treatment to 60 days of treatment for all sealers (p < 0.05). In the 3-week-old biofilm group, 60 days of exposure to bioceramic sealers resulted in more significant dead bacteria than 7-day exposures of the biofilms (p < 0.05). Bioceramic sealers were more effective in killing bacteria than the GuttaFlow 2 sealer (p < 0.05). CONCLUSIONS: Calcium silicate-based sealers showed good antimicrobial effects against biofilms within dentinal tubules, especially in the first week in young biofilms. There is no substantive antibacterial activity observed for the examined root canal sealers against young dentinal tubule biofilms. CLINICAL RELEVANCE: The bioceramic root canal sealers examined demonstrate minimal additional antibacterial effects after long-term exposure to young biofilms.

Progression of non-carious cervical lesions: 3D morphological analysis.

OBJECTIVES: This longitudinal study aimed to investigate morphologically and quantitatively the progression of non-carious cervical lesions (NCCLs) using a confocal laser scanning microscope (CLSM) and replica models. MATERIALS AND METHODS: The samples examined comprised sets of replicas annually obtained from 83 lesions in 16 participants over 3 to 5 years. All lesions were visually categorized as wedge-shaped, saucer-shaped, or mixed-shaped lesions. CLSM images of the replicas were analyzed in terms of axial depth, occlusogingival width (height) in the buccolingual cross-section, and estimated volume using a custom code of the image analysis software to estimate the progression of the NCCLs over time. The morphological characteristics of the NCCLs were also objectively divided into three groups according to the depth to height ratio (D/H ratio). Fisher's exact test and the Cochran-Armitage trend test were used for statistical analysis. RESULTS: Saucer-shaped lesions progressed mainly in height, whereas wedge-shaped lesions increased both in height and depth. Annual progression in depth and volume significantly increased as the D/H ratio increased. More than half of the NCCLs with a small D/H ratio progressed 50 µm or more in height, whereas none of them progressed more than 50 µm in depth. Annual progression in depth significantly increased as the lesion depth at baseline increased. CONCLUSIONS: Progression patterns significantly differed between NCCLs of different shapes. Most NCCLs progressed slowly in depth regardless of their shape. Moreover, NCCLs may progress through active and inactive stages.

In vitro antibacterial effects of photodynamic therapy against Enterococcus faecalis in root canals of deciduous teeth.

OBJECTIVE: This study aimed at evaluating the in vitro antibacterial efficacy of photodynamic therapy (PDT) on planktonic E. faecalis and its biofilm in the root canal of infected deciduous teeth. METHODS: Forty root canals of maxillary deciduous anterior teeth were enlarged up to #35 K-file and inoculated with E. faecalis for 21 days. The root canals were randomly assigned into four groups (n = 10): The normal saline group (control), 1% NaClO group, PDT group, and the 1% NaClO + PDT group. Paper point samples were obtained at baseline (S1) and after treatment (S2). The colony-forming units (CFU) were counted, and the bacterial growth rate calculated. From each subgroup, 5 samples were randomly selected after treatment and a scanning laser confocal microscope (CLSM) used to determine the distribution of dead / living bacteria on the biofilm surface of each subgroup. A scanning electron microscope (SEM) was used to observe bacterial morphologies in the root canal walls of the remaining 5 samples in each subgroup. The Kruskal-Wallis test and Dunn test with boferroni adjustment were used to analyze the effect of the different treatment techniques on the E. faecalis in root canals. RESULTS: Compared to the saline group, PDT significantly reduced bacterial counts in the root canal (p < 0.05). The CFU counts were lowest (p < 0.05) in the 1% NaClO and in 1% NaClO + PDT groups. The rate of bacterial death on the surface of the biofilm in the PDT group was significantly increased after treatment (p < 0.05), and the rate of bacterial death was highest in 1%NaClO group and 1%NaClO + PDT group (p < 0.05). CONCLUSION: PDT has an antibacterial activity against E. faecalis in the root canal of deciduous teeth. Its activity against planktonic E. faecalis is better than the activity on the intact biofilm. The antibacterial activity of PDT on E. faecalis in root canals of deciduous teeth is lower compared to that of 1% NaClO.

Can the hydrogel form of sodium ascorbate be used to reverse compromised resin infiltrant penetration after bleaching?

Aims: The aim of this study was to investigate the effects of an antioxidant on the bleaching-induced reduction in the penetration depth of infiltrant resins. Materials and Methods: White spot lesions (WSLs) were created on 105 bovine tooth samples, each measuring 6 × 4 × 4 mm. Five samples were randomly selected for the examination of lesion characteristics. The remaining 100 samples were then divided into four groups (n = 25). In Group I, the WSLs were treated with resin infiltration (RI) only. RI was performed on Group II immediately after bleaching. In Group III, an antioxidant was applied for 2 h after bleaching, and this was immediately followed by RI. The Group IV samples were treated with RI at the end of a 1-week waiting period after bleaching. The penetration depths were evaluated through confocal laser scanning microscopy. Results: The lowest penetration rate, which was approximately 57%, was observed in Group II. This was followed by Group III (87%), Group IV (90%), and Group I (92%). Group II, in which the samples were infiltrated immediately after bleaching, had the lowest mean penetration percentage. All the bleached groups exhibited significantly lower penetration percentages than the nonbleached group (Group I) (P < 0.05). Antioxidant application increased the penetration significantly (P < 0.05). Conclusion: Application of sodium ascorbate was found to reverse the reduced resin penetration depth and penetration percentages resulting from bleaching. The postponement of adhesive procedures after bleaching yielded similar results.

Aberration on excitation focal spot caused by oblique interface with refractive indices discontinuous and its correction with pure-phase compensation for laser scanning microscopy.

The interface of mediums with refractive indices discontinuous, for example air-glass and glass-water, are inevitable in microscopic imaging. In this work, the aberration of oblique interface with refractive index discontinuous on the laser scanning microscope was investigated theoretically with numerical simulations. It was found that the position, shape and FWHM of focal spots, were all significantly affected by the aberration due to oblique interface. The aberration can cause serious shifting of focal spots in the axial direction of beam during z -scanning and lead to an inaccurate reconstruction of three-dimensional (3D) targets. The aberration can also lead to a decreasing spatial resolution. To correct the influence of the aberration, a pure-phase modulation method has been proposed. By applying a phase compensation map into a spatial light modulator (SLM), the oblique interface aberration had been corrected experimentally in a laser scanning microscope. We hope this research can attract the attention of researchers when using scanning microscope, especially for reconstructing 3D biological and material structures.

The protective effects of polyamines on salinity stress tolerance in foxtail millet (Setaria italica L.), an important C4 model crop.

ABSTRACT: Soil salinity is a major abiotic stress that adversely affects crop growth, development and productivity worldwide. In this study, the individual and synergistic roles of putrescine (Put) and spermidine (Spd) in salinity stress tolerance of foxtail millet (Setaria italica L.) was assessed. In the present study, plants treated with combined biogenic amines Put + Spd possess very efficient antioxidant enzyme systems which help to control the uninhibited oxidation and protect the plants from oxidative damage by ROS scavenging. Additionally, lower concentration of Put + Spd under NaCl stress showed reduced hydrogen peroxide, electrolyte leakage and caspase-like activity than control. FTIR analysis underlying the ability of PAs induced tolerance and the chemical bonds of Put + Spd treated plants were reminiscent of control plants. Moreover, histochemical analysis with 2',7'-dichlorofluorescein diacetate (DCF-DA), 3,3'-Diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) revealed that ROS accumulation was inhibited by combined PAs under salt stress condition. These results showed that Put + Spd significantly improve the endogenous PAs, which enhance high-salinity stress tolerance by detoxifying ROS. For the first time, the synergistic ROS scavenging ability of Put along with Spd was investigated upon salinity tolerance in C4 model foxtail millet crop. Overall, our findings illustrated the implication for improving salinity tolerance of agronomically important crop species.

Comparative Evaluation of Antifungal Activity of Octenidine: An In Vitro Confocal Laser Study.

AIM: The aim of this study was to evaluate the antifungal efficacy of a novel endodontic irrigant octenidine against conventional irrigants sodium hypochlorite and EDTA on Candida albicans growth in the young and old population by calculating the number of colonies formed and by qualitative identification of dead/viable fungi by Confocal laser scanning microscopy (CLSM) method. MATERIALS AND METHODS: The total number of samples used in the study was eighty samples after decoronation of the crown portion the middle third of root canal. Each group was subdivided into four subgroups with various irrigation protocols: (A) 17% EDTA + 5.25% NaOCl, (B) 100% Octenisept, (C) 17% EDTA + 5.25% NaOCl + 1% clotrimazole, and (D) phosphate buffer saline. After completion of irrigation ATCC samples (90028) of C. albicans were inoculated with 5 mL of peptone water each and incubated at 37°C for 72 hours to attain the turbidity corresponding to 0.5 McFarland standards CFU. Eight samples were analyzed for the formation of candidal colonies, and two samples for the assessment of viability of Candida by confocal laser scanning microscope in each subgroup. RESULTS: Comparison of antifungal efficacy of endodontic irrigants employed in the young and old populations revealed a significant reduction in the mean values of CFU and the mean values of percentage of non-viable microorganism by CLSM method. A positive relationship was revealed in the younger population which had a better antifungal efficacy than the older population in all the irrigant subgroups evaluated in the study. CONCLUSION: All the endodontic irrigants employed in our study had a good antifungal efficacy against Candida albicans. Octenisept had a maximum antifungal efficacy, while phosphate saline showed the least efficacy in both age groups, which was quantitatively evaluated by CFU method, and the same was confirmed through qualitative evaluation by CLSM method. CLINICAL SIGNIFICANCE: Candida albicans plays a major role in the establishment and pathogenesis of failed root canal treatment. Age-related changes alter the adhesion potential of dentin, in turn influence the outcome of endodontic therapy. Octenidine, a novel antifungal agent, can be substituted over the conventionally used EDTA and NaOCl with less adverse effects.

Comparative Efficacy of Resin Infiltrant and Two Remineralizing Agents on Demineralized Enamel: An In Vitro Study.

AIM: To compare and evaluate the caries preventive effectiveness of resin infiltrant (ICON), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (GC Tooth Mousse), and nanohydroxyapatite (Aclaim) on incipient enamel lesions. MATERIALS AND METHODS: A total of 60 human maxillary incisors extracted for periodontal reasons were included in this study. The sectioning was done at the middle third region of the crown for the 60 samples with approximate dimensions of 5 × 5 × 5 mm). In order to create the artificial enamel lesions, the samples were demineralized by placing in a beaker containing the prepared demineralizing solution for 14 days. The study samples were then divided into four groups that are resin infiltrant (group I), CPP-ACP (group II), nanohydroxyapatite (group III), and control (group IV) with 15 enamel samples in each group. The caries preventive efficacy of each group was evaluated using a confocal laser scanning microscope. RESULTS: The mean values after demineralization of enamel samples in demineralizing solution are 245 µm for resin infiltrant (group I), 246 µm for CPP-ACP (group II), 250 µm for nanohydroxyapatite (group III), and 247 µm for control (group IV). After remineralizing the enamel samples for a period of 30 days, the results are group I (resin infiltrant) 158 µm > group II (CPP-ACP) 28.8µm ≥ group III (nanohydroxyapatite) 26.3 µm. After subjecting it to demineralizing solution again for 14 days, the amount of material that was resistant to acid attack was group I (resin infiltrant) 114 µm (72%) > group III (CPP-ACP) 16.4 µm (57%) ≥ group III (nanohydroxyapatite) 13.8 µm (50%). The untreated control group showed increased progression of lesion and least resistance to acid challenge. CONCLUSION: Based on the results from this in vitro study, it can be concluded that when compared to the two remineralizing agents the resin infiltrant showed better caries preventive effectiveness. CLINICAL SIGNIFICANCE: Resin infiltrants have a favorable penetration potential in subsurface or incipient enamel lesions.

Introducing BAIT (Biofilm Architecture Inference Tool): a software program to evaluate the architecture of oral multi-species biofilms.

Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many in vitro biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate ('placebo') or stannous fluoride (SnF2). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF2 (1000, 3439 or 10 000 p.p.m. Sn2+) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn2+ treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (P<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

Adaptive mechanism of Acidithiobacillus thiooxidans CCTCC M 2012104 under stress during bioleaching of low-grade chalcopyrite based on physiological and comparative transcriptomic analysis.

Acidithiobacillus thiooxidans (A. thiooxidans) is often used for sulfur-bearing ores bioleaching, but its adaptive mechanism to harsh environments remains unclear. Here, we explored the adaptive mechanism of A. thiooxidans in the process of low-grade chalcopyrite bioleaching based on the physiology and comparative transcriptome analysis. It was indicated that A. thiooxidans maintains intracellular pH homeostasis by regulating unsaturated fatty acids, especially cyclopropane fatty acids, intracellular ATP, amino acid metabolism, and antioxidant factors. Comparative transcriptome analysis indicated that the key genes involved in sulfur oxidation, sor and soxABXYZ, were significantly up-regulated, generating more energy to resist extreme environmental stress by more active sulfur metabolism. Confocal laser scanning microscope analysis found that down-regulation of flagellar-related genes was likely to promote the biofilm formation. System-level understanding of leaching microorganisms under extreme stress can contribute to the evolution of these extremophiles via genetic engineering modification work, which further improves bioleaching in future.

Evaluation of the Antibacterial Efficiency of a Combination of 1% Alexidine and Sodium Hypochlorite on Enterococcus faecalis Biofilm Models: An In Vitro Study.

AIM: The aim of the study was to assess the antibacterial efficiency of a combination of 1% alexidine (ALX) and 5.25% sodium hypochlorite (NaOCl) against E. faecalis biofilm using a confocal scanning electron microscopy. MATERIALS AND METHODS: An estimated 120 human root dentin disks were prepared, sterilized, and inoculated with E. faecalis strain (ATCC 29212) to develop a 3-weeks-old biofilm. The dentin discs were exposed to group I-control group: 5.25% sodium hypochlorite (NaOCl) (n = 20); group II-1% ALX + 5.25% NaOCl (n = 40); group III-1% alexidine (ALX) (n = 40) (Sigma-Aldrich, Mumbai, India); group IV-negative control: saline (n = 20). After exposure, the dentin disks were stained with the fluorescent live/dead dye and evaluated with a confocal scanning electron microscope to calculate the proportion of dead cells. Statistical analysis was done using the Kruskal-Wallis and Mann-Whitney U test (p < 0.05). RESULTS: The maximum proportion of dead cells were seen in the groups treated with the combination of 1% ALX + 5.25% NaOCl (94.89%) and in the control group 5.25% NaOCl (93.14%). The proportion of dead cells presented in the 1% ALX group (51.79%) and negative control group saline (15.10%) were comparatively less. CONCLUSION: The antibacterial efficiency of a combination of 1% ALX and 5.25% NaOCl was more effective when compared with 1% ALX alone. CLINICAL SIGNIFICANCE: Alexidine at 1% could be used as an alternative endodontic irrigant to chlorhexidine, as alexidine does not form any toxic precipitates with sodium hypochlorite. The disinfection regimen comprising a combination of 1% ALX and 5.25% NaOCl is effective in eliminating E. faecalis biofilms.

Proteomics analysis of crude squid ink isolated from Sepia esculenta for their antimicrobial, antibiofilm and cytotoxic properties.

The present study deals with the proteomics analysis of crude squid ink isolated from Sepia esculenta for their antibacterial, antifungal, antibiofilm and cytotoxic properties. To achieve this, SDS-PAGE was used to separate proteins as bands, In-gel trypsin digested and analyzed by MALDI-TOF mass spectrometry. A total of 4 bands were identified by MASCOT search analysis namely astacin-like squid metalloprotease type I (ASMT-I), 70 kDa neurofilament protein (NP), uncharacterized protein LOC106181966 isoform X1 (UP-Iso-X1) and Ommochrome-binding protein (Oc-BP). Further, the obtained crude squid proteins were subjected to antimicrobial and antibiofilm activities against pathogenic bacterial and fungal strains respectively. Further, MTT assay was also carried out to deliberately explain the cytotoxic ability of crude squid ink protein against MCF-7 breast cancer cell lines. The results from the study revealed that, the proteins are shown to be toxic against pathogenic strains and breast cancer cell lines in a dose-dependent manner. More importantly, the proteins are well enough to eradicate biofilms substantiated by light and confocal laser scanning microscopic observations. Altogether, the crude squid ink proteins hampered the growth of breast cancer cells with an IC50 value of 65.3 ±â€¯0.46 µg mL-1. In conclusion, it is believed that the proteins from crude squid ink will provide new insights in hampering bacterial biofilms and cancer in near future.

Antibiofilm effect of Nocardiopsis sp. GRG 1 (KT235640) compound against biofilm forming Gram negative bacteria on UTIs.

Urinary tract infections (UTIs) are diverse public health complication and caused by range of pathogens, however mostly Gram negative bacteria cause significant life threatening risks to different populations. The prevalence rate and antimicrobial resistance among the Gram negative uropathogens alarmed significantly heighten the economic burden of these infections. In this study, we investigated the antibiofilm efficiency of Pyrrolo [1,2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) extracted from endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) against P. mirabilis and E. coli. The extracted compound was characterized through TLC, HPLC, GC-MS, LC-MS and confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM). The compound, Pyrrolo [1,2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) inhibits both bacterial biofilm formation as well as reduces the viability of preformed biofilms. Furthermore, CLSM image shows cell shrinkage, disorganized cell membrane and loss of viability. The SEM result also confirms the cell wall degradation in treated cells of the bacteria. Hence, the Pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) is active against P. mirabilis and E. coli.

Biofilm diversity, structure and matrix seasonality in a full-scale cooling tower.

Biofilms commonly colonise cooling water systems, causing equipment damage and interference with the operational requirements of the systems. In this study, next-generation sequencing (NGS), catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH), lectin staining and microscopy were used to evaluate temporal dynamics in the diversity and structure of biofilms collected seasonally over one year from an open full-scale cooling tower. Water samples were analysed to evaluate the contribution of the suspended microorganisms to the biofilm composition and structure. Alphaproteobacteria dominated the biofilm communities along with Beta- and Gammaproteobacteria. The phototrophic components were mainly cyanobacteria, diatoms and green algae. Bacterial biodiversity decreased from winter to autumn, concurrently with an increase in cyanobacterial and microalgal richness. Differences in structure, spatial organisation and glycoconjugates were observed among assemblages during the year. Overall, microbial variation appeared to be mostly affected by irradiance and water temperature rather than the source of the communities. Variations in biofilms over seasons should be evaluated to develop specific control strategies.

Wear profile of canal wall surfaces and bond strength of endodontic sealers after in situ acid challenge.

AIM: To evaluate the wear of root wall surfaces, the bond strength of sealers to dentine and the demineralization around root filling materials after canals were exposed to acid challenge in situ. METHODOLOGY: Eighty-seven roots of mandibular incisors were selected. Thirty-two were used in the laboratory bond strength study (n = 8), and 55 in the in situ study (n = 11). Root canals were prepared biomechanically and then filled with gutta-percha and AH Plus, MTA Fillapex, Sealapex or Endofill. For 14 days, 11 participants used intra-oral devices with five sterilized roots (four experimental and one control - only canal prepared). Drops of sucrose were dripped onto roots allowing the accumulation of biofilm on canal surfaces. Roots were removed, sectioned and analysed for the following: bond strength of filling material using a push-out test and also wear profile and dentine demineralization using confocal microscopy. Bond strength (MPa) was evaluated by two-way anova and Tukey test (α = 0.05), and wear profile was assessed by Kruskal-Wallis and t-tests (α = 0.05). RESULTS: AH Plus had the highest bond strength values. Intermediate results were found in roots with MTA Fillapex and Endofill, whilst Sealapex had inferior results (P < 0.05). No significant differences were found amongst root thirds (P > 0.05). For wear profile, samples were associated with degradation of the filling materials after exposure to the oral environment (P < 0.05). Roots had signs of demineralization around the filling material when Sealapex and Endofill were used. CONCLUSIONS: Sealers were not able to prevent degradation of the adhesive interface and dentine. AH Plus and MTA Fillapex had superior bond strength to dentine and less intense demineralization around the root filling.

Confocal laser scanning microscopy-a powerful tool in bone research.

The confocal laser scanning microscope (CLSM) enables the collection of images picturing selected planes in depth of thick samples, thus giving 3D information while keeping the sample intact. In this article we give an overview of our CLSM applications in bone research: (i) the characterization of osteoblasts and osteoclasts properties in cell biology, (ii) the visualization of the three dimensional (3D) osteocyte lacunar canalicular network in undemineralized plastic-embedded bone samples, (iii) the observation of tetracycline labels in bone biopsy samples from patients in combination with information on the mineralization density from quantitative backscatter electron imaging, which enables the time course of mineral accumulation in newly formed bone to be followed, (iv) the precise measurement of the thickness of thin ground bone sections, a prerequisite for the mapping of local mechanical properties by scanning acoustic microscopy.

A novel quantitative volumetric spreading index definition and assessment of astrocyte spreading in vitro.

A novel quantitative volumetric spreading index (VSI) is defined that depends on the total distance between object voxels and the contact surface plane in three-dimensional (3D) space. The VSI, which ranges from 0 to 1, is rotationally invariant around the z-axis. VSI can be used to quantify the degree of individual cell spreading, which is important for analysis of cell interactions with their environment. The VSIs of astrocytes cultured on a nanofibrillar surface and three different comparative planar surfaces have been calculated from confocal laser scanning microscope z-series images, and the effects of both culture surface and immunoreactivity on the degree of cell spreading were investigated. VSI calculations indicated a statistical correlation between increased reactivity, based on immunolabeling for glial fibrillary acidic protein, and decreased cell spreading. Further results provided a quantitative measure for the increased spreading of quiescent-like and reactive-like astrocytes on planar substrates functionalized with poly-l-lysine. © 2017 International Society for Advancement of Cytometry.

In vivo Imaging of the Cerebral Endothelial Glycocalyx in Mice.

BACKGROUND/AIMS: Endothelial glycocalyx refers to the proteoglycan or glycoprotein layer of vessel walls and has critical physiological functions. Cerebral glycocalyx may have additional functions considering the blood-brain barrier and other features. However, the assessment of it has only been performed ex vivo, which includes processes presumably damaging the glycocalyx layer. Here we visualize and characterize the cerebral endothelial glycocalyx in vivo. METHODS: We visualized and quantified the cerebral endothelial glycocalyx in vivo under a 2-photon microscope by tagging glycocalyx and vessel lumen with wheat germ agglutinin lectin and dextran, respectively. The radial intensity was analyzed to measure the thickness of the cerebral endothelial glycocalyx in each vessel type. RESULTS: Cerebral arteries and capillaries have an intact endothelial glycocalyx, but veins and venules do not. The thickness of the glycocalyx layer in pial arteries, penetrating arteries, and capillaries was different; however, it was not correlated with the vessel diameter within each vessel type. CONCLUSION: We characterized the distribution of the cerebral endothelial glycocalyx in vivo. Compared to the results from ex vivo studies, the layer is thicker, indicating that the layer may be damaged in ex vivo systems. We also observed an inhomogeneous cerebral endothelial glycocalyx distribution that might reflect the functional heterogeneity of the vessel type.