Confocal Laser Scanning Microscopy Analysis Research Articles

Compact and water flushing resistant mesh biofilms forming at short SRT disappeared naturally under extended SRT in dynamic membrane bioreactor

Extending the solids retention time (SRT) has been demonstrated to mitigate membrane biofouling. Nevertheless, it remains an intriguing question whether the compact and water flushing resistant mesh biofilms developed at short SRT can undergo biodegradation and be removed with extended SRT. In present study, the bio-fouled mesh filter in the 10d-SRT dynamic membrane bioreactor (DMBR), with mesh surfaces and pores covered by compact and water flushing resistant biofilms exhibiting low water permeability, was reused in the 40d-SRT DMBR without any cleanings. After being reused at 40d-SRT, its flux driven by gravity occurred from the 10th day and recovered to a regular level of 36.7 L m−2·h−1 on the 27th day. Both scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) analyses indicated that the compact mesh biofilms formed at10d-SRT biodegraded and were removed at 40d-SRT, with the residual biofilms becoming removable by water flushing. As a result, the hydraulic resistance of the bio-fouled mesh filter decreased from 4.36 × 108 to 6.97 × 107 m−1, and its flux fully recovered. The protein and polysaccharides densities in mesh-biofilms decreased from 24.4 to 9.7 mg/cm2 and from 10.7 to 0.10 mg/cm2, respectively, which probably have contributed to the disappearance of compact biofilms and the decrease in adhesion. Furthermore, the sludge and mesh-biofilms in the 40d-SRT reactor contained a higher relative abundance of dominant quorum quenching bacteria, such as Rhizobium (3.52% and 1.35%), compared to those in the 10d-SRT sludge (0.096%) and mesh biofilms (0.79%), which might have been linked to a decline in extracellular polymeric substances and, consequently, the biodegradation and disappearance of compact biofilms.

An in vitro evaluation of Icon resin infiltrant penetration into demineralized enamel lesions using an indirect staining technique with confocal laser scanning microscope analysis in dual fluorescence mode

Abstract Context: White spot lesion is the first clinical sign of a caries lesion and represents mineral loss from the enamel subsurface. Aim: The aim of this study was to evaluate the penetration depth (PD) of Icon resin infiltrant into artificially demineralized enamel lesions using confocal laser scanning microscope (CLSM) analysis in dual fluorescence mode. Settings and Designs: The design of the study was an in vitro study. Materials and Methods: 22 extracted human permanent maxillary central incisor teeth were collected, and enamel sections were obtained from the coronal middle third. All enamel specimens were exposed to demineralization and remineralization solutions for 14 days. On positive confirmation of enamel demineralization by scanning electron microscope analysis, 20 specimens were then subjected to Icon resin infiltration following manufacturer instructions. Specimens were processed with indirect staining technique using rhodamine B and sodium fluorescein dyes and examined under CLSM at ×10 magnification in dual fluorescence mode using ImageJ software to evaluate PD of resin infiltrant into demineralized enamel lesions. Statistical Analysis Used: Obtained data were analyzed using an independent t-test. P ≤0.05 was considered statistically significant. Results: The maximum depth of demineralized enamel lesion was 590 μm, and the mean depth was 290.78 ± 14.80 μm. The maximum depth of resin infiltrant penetration was 580 μm, and the mean depth was 279.08 ± 13.88 μm; P = 0.006. The percentage penetration of resin infiltrant was 95.99%. Conclusion: Icon resin infiltrant was highly effective in its depth of penetration into demineralized enamel lesions. The use of indirect staining and CLSM analysis in dual fluorescence mode is more reliable and accurate technique to evaluate the PD of resin infiltrant.

Abstract 7502: MCT1 and ΜCT4 on circulating tumor cells isolated from non small cell lung cancer patients

Abstract Background: Lung cancer is the leading cause of cancer-related deaths. Non-small-cell lung cancer (NSCLC) is the most common subtype of lung cancer and is characterized by poor prognosis and survival rate. This is associated with the fact that most patients are diagnosed at advanced stages of the disease and the possibility of recurrence after treatment remains high. Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) play a crucial role in cellular metabolism, as they are responsible for the transport of lactate and other monocarboxylic acids across the plasma membrane. Their function can further regulate cell survival and proliferation, especially under hypoxic conditions. To elucidate the relationship between CTCs and the metabolic aspect of cancer, we aimed to investigate the expression of MCT1 and MCT4 in CTCs derived from NSCLC patients and its potential association with patients’ outcome. Methods: Fifty-three NSCLC patients at baseline were enrolled in the current study. CTCs were isolated using the ISET system and triple immunofluorescence experiments were performed, using CK, MCT1, MCT4, and CD45 antibodies. The expression of MCT1 and MCT4 in CTCs was assessed by confocal laser scanning microscopy and statistical analysis was performed using SPSS software. Results: CTCs were detected in 29 out of 53 (55%) NSCLC patients. CK expression was characterized as high or low, with the predominant phenotypes among CK-positive patients being the CKhigh/MCT1+/CD45-and CKlow/MCT4-/CD45- (63% and 66%, respectively). Whereas the phenotype CKhigh/MCT4+/CD45- was detected in 52% of the patients. The phenotypes with the lowest frequency were the CKlow/MCT4+/CD45- (7%), CKlow/MCT1+/CD45- (15%), and the CKhigh/MCT1-/CD45- (15%). Most of the isolated CTCs had the CKhigh/MCT1+/CD45- and CKlow/MCT4-/CD45- phenotypes with a mean percentage of 48% and 51% respectively, while only a small number of CTCs per patient were CKlow/MCT1+/CD45- or CKlow/MCT4+/CD45- (8%and 5%, respectively). The phenotype CKlow/MCT1-/CD45- was associated with poorer overall survival (OS) [p = 0.005, (15 vs 33 months respectively)], HR=0.351), while the presence of more than 3 CKhigh/MCT4+/CD45- CTCs was related to poorer progression-free survival (PFS) [p = 0.042 (4 vs 17 months respectively)]. Conclusions: MCT1 and MCT4 were expressed in a high proportion of NSCLC patients at baseline and may constitute interesting biomarkers relevant to cancer progression. However, further analysis in a larger cohort of patients is required to clarify their potential significance in clinical practice. Citation Format: Karolina Mangani, Evangelia Pantazaka, Evi Lianidou, Vassilis Gerogoulias, Athanasios Kotsakis, Athina Markou, Galaktia Kallergi. MCT1 and ΜCT4 on circulating tumor cells isolated from non small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7502.

Antilisterial Effectiveness of Origanum vulgare var. hirtum and Coridothymus capitatus Essential Oils and Hydrolates Alone and in Combination.

The antimicrobial activity of Origanum vulgare var. hirtum (O) and Coridothymus capitatus (C) essential oils (EOs) and hydrolates (HYs) of the same botanical species was evaluated on sixteen L. monocytogenes strains from food and clinical origins. The antimicrobial activity was assessed by Minimum Inhibitory Concentration (MIC) determination, viable cell enumeration over time up to 60 min, and evaluation of the cellular damage through Confocal Laser Scanning Microscope (CLSM) analysis. EOs exhibited antimicrobial activity with MIC values ranging from 0.3125 to 10 µL/mL. In contrast, HYs demonstrated antimicrobial effectiveness at higher concentrations (125-500 µL/mL). The effect of HYs was rapid after the contact with the cells, and the cell count reduction over 60 min of HY treatment was about 1.2-1.7 Log CFU/mL. L. monocytogenes cells were stressed by HY treatment, and red cell aggregates were revealed through CLSM observation. Moreover, the combinations of EOs and HYs had an additive antilisterial effect in most cases and allowed the concentration of use to be reduced, while maintaining or improving the antimicrobial effectiveness. The combined use of EOs and HYs can offer novel opportunities for applications, thereby enhancing the antimicrobial effectiveness and diminishing the concentration of use. This provides the added benefit of reducing toxicity and mitigating any undesirable sensory effects.

Genotype-phenotype evaluation of the heterogeneity in biofilm formation by diverse Bacillus licheniformis strains isolated from dairy products

The spoilage bacterium Bacillus licheniformis has been identified as a quick and strong biofilm former in the dairy industry. In our previous study, intra-species variation in bacterial biofilms has been observed in diverse B. licheniformis strains from different genetic backgrounds; however, the mechanisms driving the observed heterogeneity of biofilms remain to be determined. In this study, the genotype-phenotype evaluation of the heterogeneity in biofilm formation of four B. licheniformis strains were examined. The heterogeneity in biofilm phenotype was accessed in aspects of bacterial growth and motility, cell viability, biofilm matrix production, and biofilm architectures. The underlying mechanisms of the intra-species variability in biofilms were also explored by whole genome resequencing (WGR). Results from bacterial motility tests showed a diverse motility among the strains, but there was no clear correlation between bacterial motility and biofilm formation. The cell viability results showed a different number of live cells in biofilms at the intra-species level. Analysis of chemical components in biofilm matrix demonstrated the great intra-species differences regarding extracellular matrix composition, and a negative correlation between biofilm formation on stainless steel and the protein: carbohydrate ratio in biofilm matrix was observed. Confocal laser scanning microscopy analysis also revealed the intra-species variability by showing great differences in general properties of B. licheniformis biofilms. WGR results identified important pathways involved in biofilm formation, such as two-component systems, quorum sensing, starch and sucrose metabolism, ABC transporters, glyoxylate and dicarboxylate metabolism, purine metabolism, and a phosphotransferase system. Overall, the above results emphasize the necessity of exploring the intra-species variation in biofilms, and would provide in-depth knowledge for designing efficient biofilm control strategies in the dairy industry.

Role of Selenoprotein W in participating in the progression of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide. Numerous evidence has demonstrated that metabolic reprogramming serves as a hallmark associated with an elevated risk of NAFLD progression. Selenoprotein W (SelW) is an extensively expressed hepatic selenoprotein that plays a crucial role in antioxidant function. Here, we first demonstrated that SelW is a significantly distinct factor in the liver tissue of NAFLD patients through the Gene Expression Omnibus (GEO) database. Additionally, loss of SelW alleviated hepatic steatosis induced by a high-fat diet (HFD), and was accompanied by the regulation of metabolic and inflammatory pathways as verified by transcriptomic analysis. Moreover, co-immunoprecipitation (CO-IP), liquid chromatography-tandem mass spectrometry (LC-MS), laser scanning confocal microscopy (LSCM) and molecular docking analysis were subsequently implemented to identify Pyruvate Kinase M2 (PKM2) as a potential interacting protein of SelW. Meanwhile, SelW modulated PKM2 translocation into the nucleus to trigger transactivation of the HIF-1α, in further mediating mitochondrial apoptosis, eventually resulting in mitochondrial damage, ROS excessive production and mtDNA leakage. Additionally, mito-ROS accumulation induced the activation of the NLRP3 inflammasome-mediated pyroptosis, thereby facilitating extracellular leakage of mtDNA. The escaped mtDNA then evokes the cGAS-STING signaling pathway in macrophage, thus inducing a shift in macrophage phenotype. Together, our results suggest SelW promotes hepatocyte apoptosis and pyroptosis by regulating metabolic reprogramming to activate cGAS/STING signaling of macrophages, thereby exacerbating the progression of NAFLD.

Alzheimer's Disease-associated Region-specific Decrease of Vesicular Glutamate Transporter Immunoreactivity in the Medial Temporal Lobe and Superior Temporal Gyrus

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets.

Modifying gas transfer membranes with nanoscale zero-valent iron: effects on membrane material properties, treatment performance, and biofilm thickness.

Excessive membrane biofilm growth on membrane fibers depends on various factors, with membrane properties playing a pivotal role in influencing microbial affinity for the membrane. To investigate the antibacterial impact of nano-sized zero-valent iron (nZVI) on membrane biofilm structure, pristine (polyvinylidene fluoride (PVDF)) only: HF-0 (PVDF:20/nZVI:0 w/w) and four gas transfer membranes (PVDF:nZVI at different concentrations: HF-1 (PVDF:20/nZVI:0.25 w/w), HF-2 (PVDF:20/nZVI:0.50 w/w), HF-3 (PVDF:20/nZVI:0.75 w/w), HF-4 (PVDF:20/nZVI:1.0 w/w)) were produced. These membranes were assessed for surface morphology, porosity, gas permeability, and biofilm thickness, which ultimately affect biochemical reaction rates in membrane biofilm reactors (MBfRs). Various MBfRs utilizing these gas transfer membranes were operated at different hydraulic retention times (HRTs) and oxygen pressures to assess chemical oxygen demand (COD) removal efficiency and nitrification performance. Incorporating nZVI into the PVDF polymer solution increased surface hydrophilicity and porosity but reduced Young's Modulus and oxygen diffusion coefficients. Confocal laser scanning microscopy (CLSM) analysis revealed an average biofilm thickness of 700 μm in HF-0, HF-1, and HF-3, with a 100 μm decrease in HF-2, even though Escherichia coli growth was observed in HF-3 fibers. Regardless of nZVI dosage, a significant decline in COD removal and nitrification rates occurred at low HRTs and gas pressures.

Advancing Glucose Sensing Through Auto‐Fluorescent Polymer Brushes: From Surface Design to Nano‐Arrays

AbstractDesigning smart (bio)interfaces with the capability to sense and react to changes in local environments offers intriguing possibilities for new surface‐based sensing devices and technologies. Polymer brushes make ideal materials to design such adaptive and responsive interfaces given their large variety of functional and structural possibilities as well as their outstanding abilities to respond to physical, chemical, and biological stimuli. Herein, a practical sensory interface for glucose detection based on auto‐fluorescent polymer brushes decorated with phenylboronic acid (PBA) receptors is presented. The glucose‐responsive luminescent surfaces, which are capable of translating conformational transitions triggered by pH variations and binding events into fluorescent readouts without the need for fluorescent dyes, are grown from both nanopatterned and non‐patterned substrates. Two‐photon laser scanning confocal microscopy and atomic force microscopy (AFM) analyses reveal the relationship between the brush conformation and glucose concentration and confirm that the phenylboronic acid functionalized brushes can bind glucose over a range of physiologically relevant concentrations in a reversible manner. The combination of auto‐fluorescent polymer brushes with synthetic receptors presents a promising avenue for designing innovative and robust sensing systems, which are essential for various biomedical applications, among other uses.

Analysis of antibiofilm activity gold and silver nanoparticles against uropathogenic Escherichia coli using confocal laser scanning microscopy

Infectious diseases, including urinary tract infections (UTIs), significantly impact global health, diminishing the quality of life and increasing clinical and economic burdens. Concerns arise due to the potential development of antimicrobial resistance (AMR) facilitated by biofilm formation. Nanotechnology, specifically silver and silver nanoparticles (1–100 nm), proves effective against bacterial strains, making them valuable in the biomedical field. Advances in microscopic imaging techniques, such as Confocal Laser Scanning Microscopy (CLSM), enhance our understanding of biofilms. This study aims to assess the effectiveness and optimal concentration of gold and silver nanoparticles in combating biofilms formed by uropathogenic Escherichia coli (UPEC) through CLSM analysis. The research comprises the following stages: rejuvenation of bacterial isolates and inoculum preparation, testing the anti-biofilm activity of gold and silver nanoparticles at concentrations of 25, 50, 75, and 100 ppm against UPEC using CLSM analysis. The findings reveal that both gold and silver nanoparticles exhibit anti-biofilm activity against UPEC at concentrations of 25, 50, 75, and 100 ppm, as confirmed by CLSM analysis. The optimal concentration for anti-biofilm activity of gold and silver nanoparticles against UPEC is identified as 100 ppm.

Rapid statistical discrimination of fluorescence images of multiple foodborne pathogens using multicolor-emissive carbon dots

The rapid discrimination of multiple foodborne pathogens is vital and challengeable in ensuring food safety. Herein, a facile fluorescence method was developed to distinguish different bacteria using multicolor-emissive carbon dots (CDs) based on confocal laser scanning microscopy (CLSM) and linear discriminant analysis (LDA). The array employs single CDs as receptors and indicators, two pH solutions and three excitation channels as sensing elements for multicolor imaging and recognition of multiple bacteria. Through one-step staining, the CDs were capable of rapid adsorbing onto all bacteria in 1 min with varying affinities. The discrimination ability of the array was markedly enhanced by means of the synergistic recognition of bacteria and CDs across diverse pH levels and three excitation channels. By using LDA, six kinds of bacteria could be well discriminated with the classification accuracy of 100%. Meanwhile, the feasibility has been successfully expanded to mixture and food samples discrimination.

Characterization of Salmonella ser. Enteritidis-specific bacteriophages and biocontrol strategy to reduce S. Enteritidis on egg products using bacteriophages and essential oil compounds

Salmonella contamination of egg products is a major concern in the poultry industry. This study aimed to characterize newly isolated lytic Salmonella bacteriophages and evaluate the efficacy of Salmonella ser. Enteritidis biofilm reduction using a selected phage (CAU-SEP-3 at 100 MOI), essential oil (EO) compounds (thymol [Thy] and geraniol [Ger]) at 1 × MIC, and the combination of phage and EO compounds on food-contact materials (stainless steel [SS] and polypropylene [PP]) and quail eggs (at 4 and 25 °C). On both the coupons and eggshells, the combination treatment reduced Salmonella biofilms more effectively than either of the single treatments, and no differences in texture were detected after the combination treatment on quail eggs compared with the control. Confocal laser scanning microscopy analysis and three-dimensional imaging using COMSTAT provided visualization of biofilm reduction. Gene expression levels were compared using quantitative real-time PCR analysis, indicating that the combination treatment was the most effective in reducing the avrA, hilA, rpoH, rpoS, sefA, and spvR gene expression levels. A DNAPlotter image of CAU-SEP-3 was generated based on genome sequencing data, revealing that 35 (17.6%) proteins were predicted to be functional proteins, while 147 (82.4%) were annotated as hypothetical proteins. This study demonstrates that the combination of phage and EO compounds is potentially effective in inactivating S. Enteritidis biofilms; furthermore, it may contribute to the safety of future production and processing of eggs.

Anti-Biofilm and Antivirulence Activities of 1,2,6-tri-O-galloyl-β-ᴅ-glucose against Proteus penneri.

The present study investigated the anti-virulence and anti-biofilm effects of 1,2,6-tri-O-galloyl-β-ᴅ-glucose (TGG), isolated from Camellia nitidissima Chi flowers, on Proteus penneri ALK 1200. TGG was isolated from C. nitidissima Chi flowers using various chromatographic techniques. The milk plate assay, azocasein assay, and exopolysaccharides (EPS) inhibition assay revealed that TGG effectively inhibited the production of crucial virulence factors, including protease and EPS, in P. penneri ALK 1200. Furthermore, fourier transform infrared spectroscopic (FT-IR) analysis indicated that TGG interfered with the composition of P. penneri ALK 1200's cellular component, potentially reducing the bacteria's pathogenicity. In addition, crystal violet assay, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) analysis indicated a significant reduction in biofilm formation following TGG treatment. The swimming and swarming assays also showed that TGG reduced the motility of P. penneri ALK 1200. Furthermore, the qRT-PCR assay demonstrated that TGG down-regulated the expression of positive regulatory genes (hfq and flhD) responsible for motility and biofilm formation, while up-regulating the expression of the negative regulator of the quorum sensing system, bssS, in P. penneri ALK 1200. TGG displayed potent anti-QS and anti-biofilm activity towards P. penneri ALK 1200.

Bactericidal Effect of Triple Antibiotic Paste against Enterococcus faecalis in Dentinal Tubules: An Ex Vivo Study.

The aim of this study was to investigate the bactericidal effect of various concentrations of triple antibiotic paste (TAP) against Enterococcus faecalis (E. faecalis) in dentinal tubules using a bacterial culture assay and confocal laser scanning microscope (CLSM). Ninety human teeth were contaminated with E. faecalis (ATCC 29212) and randomly allocated into 5 groups; the negative control (without TAP), 1 mg/ml, 5 mg/ml, 7.5 mg/ml and 10 mg/ml TAP (n=18). After a 3-week TAP treatment, samples were collected from the root canal space, root dentin at 100-μm and 200-μm depth. The collected samples were subjected to a bacterial culture assay (n=10). Eight roots from each group underwent CLSM analysis to determine the live/dead bacterial cells. The bacterial culture assay results indicated that the negative control samples were all culturable. The number of culture-positive samples decreased after TAP treatment at 1, 5, 7.5 and 10 mg/ml, with 2, 2, 1 and 0 culturable samples, respectively. However, there was no significant difference among the TAP treatments. Surprisingly, the CLSM analysis demonstrated live bacteria in the dentinal tubules in all samples. The negative control had 52.36%+-3.24 live bacteria. TAP treatment at 10 mg/ml had the lowest percentage of live bacterial cells (40.58%+-5.40), followed by 7.5 mg/ml (44.14%+-6.03), 5 mg/ml (46.31%+-5.32) and 1 mg/ml (52.55%+-8.82). The percentage of live cells in the 10 mg/ml, 7.5 mg/ml and 5 mg/ml TAP groups were significantly lower than the 1 mg/ml TAP and negative control groups. TAP treatment significantly decreased the percentage of viable E. faecalis cells in the dentinal tubules and its bactericidal effect was dose-dependent.

Enhancing the textural and rheological properties of fermentation-induced pea protein emulsion gels with transglutaminase.

The aim of this study was to assess how transglutaminase (TG) impacts the microstructure, texture, and rheological properties of fermentation-induced pea protein emulsion gels. Additionally, the study examined the influence of storage time on the functional properties of these gels. Fermentation-induced pea protein gels were produced in the presence or absence of TG and stored for 1, 4, 8, 12, and 16 weeks. Texture analysis, rheological measurements, moisture content and microstructure evaluation with confocal laser scanning microscopy (CLSM) and 3D image analysis were conducted to explore the effects of TG on the structural and rheological properties of the fermented samples. The porosity of the protein networks in the pea gels decreased in the presence of TG, the storage modulus increased and the textural characteristics were significantly improved, resulting in harder and more springy gels. The gel porosity increased in gels with and without TG after storage but the effect of storage on textural and rheological properties was limited, indicating limited structural rearrangement once the fermentation-induced pea protein emulsion gels are formed. Greater coalescence was observed for oil droplets within the gel matrix after 16 weeks of storage in the absence of TG, consistent with these protein structures being weaker than the more structurally stable TG-treated gels. This study shows that TG treatment is a powerful tool to enhance the textural and rheological properties of fermentation-induced pea protein emulsion gels.

Comparison of EDTA and Boric Acid Irrigation in Terms of Root Canal Dentin Microhardness and Sealer Penetration Depth

Chemo mechanical preparation is one of the important stages for successful endodontic treatment, and Boric acid has been proposed as an alternative irrigation solution for root canal treatment. Objective: The purpose of this study was to evaluate the effect of irrigation with boric acid (BA) and EDTA on the mineral content and microhardness of dentin and penetration of the AH Plus root canal sealer. Methods: 63 single-rooted teeth were instrumented. Microhardness analysis, 45 teeth were cut longitudinally and then transversely to obtain dentin. Pre- and post-treatment microhardness values were measured. The samples were randomly assigned into 3 groups (n = 15) for irrigation solution: distilled water, 17% EDTA, and 5% BA. The 18 roots were irrigated with three solutions (n = 6). Nine of the roots were filled with AH-Plus for confocal laser scanning microscopy analysis, and the others were used for XRD analysis. Results: The results were evaluated with the ANOVA test. BA caused a significant decrease in microhardness value in apical and coronal parts (p < 0.05), while EDTA reduced only in coronal parts (p < 0.05). Conclusion: BA provided higher sealer penetration ability. BA reduced the microhardness and provided better sealer penetration.

Analysis of antibiofilm activity gold and silver nanoparticles against uropathogenic Escherichia coli using confocal laser scanning microscopy

Infectious diseases, including urinary tract infections (UTIs), significantly impact global health, diminishing the quality of life and increasing clinical and economic burdens. Concerns arise due to the potential development of antimicrobial resistance (AMR) facilitated by biofilm formation. Nanotechnology, specifically silver and silver nanoparticles (1–100 nm), proves effective against bacterial strains, making them valuable in the biomedical field. Advances in microscopic imaging techniques, such as Confocal Laser Scanning Microscopy (CLSM), enhance our understanding of biofilms. This study aims to assess the effectiveness and optimal concentration of gold and silver nanoparticles in combating biofilms formed by uropathogenic Escherichia coli (UPEC) through CLSM analysis. The research comprises the following stages: rejuvenation of bacterial isolates and inoculum preparation, testing the anti-biofilm activity of gold and silver nanoparticles at concentrations of 25, 50, 75, and 100 ppm against UPEC using CLSM analysis. The findings reveal that both gold and silver nanoparticles exhibit anti-biofilm activity against UPEC at concentrations of 25, 50, 75, and 100 ppm, as confirmed by CLSM analysis. The optimal concentration for anti-biofilm activity of gold and silver nanoparticles against UPEC is identified as 100 ppm.

Dynamic in situ microstructure evaluation of cheese melting: Understanding structure-rheological relationships under controlled temperature sweeps

This study assessed the changes in processed and natural cheese during heating from 5 to 95 °C, at 2 °C per min, using confocal laser scanning microscopy (CLSM), Raman micro-spectroscopy and rheological analysis. The CLSM images showed that the cheese samples varied in fat droplet size and distribution. CLSM micrographs of processed cheese with lower fat content (<10%) showed no substantial structural changes during heating, which were consistent with the absence of cross-over in temperature rheology sweep curves. Cheese containing starch showed uniformly distributed fat and cohesive protein network, leading to reduced cheese flow. At temperatures > 70 °C, cheeses with a fat content ≥ 25% showed greater coalescence of fat globules, which finally merged into large fat pools. The results of this study demonstrate the potential of dynamic in situ CLSM and rheological analysis for evaluating the changes in cheese matrices during heating. The Raman spectral region corresponding to C-H stretching vibrations (2800–3000 cm-1) could be used to track fat melting in cheese samples with minimum 10% fat.

Biofilm formation of the food spoiler Brochothrix thermosphacta on different industrial surface materials using a biofilm reactor

Brochothrix thermosphacta is considered as a major food spoiler bacteria. This study evaluates biofilm formation by B. thermosphacta CD337(2) – a strong biofilm producer strain – on three food industry materials (polycarbonate (PC), polystyrene (PS), and stainless steel (SS)). Biofilms were continuously grown under flow at 25 °C in BHI broth in a modified CDC biofilm reactor. Bacterial cells were enumerated by plate counting, and biofilm spatial organization was deciphered by combining confocal laser scanning microscopy and image analysis. The biofilms had the same growth kinetics on all three materials and reach 8log CFU/cm2 as maximal concentration. Highly structured biofilms were observed on PC and PS, but less structured ones on SS. This difference was confirmed by structural quantification analysis using the image analysis software tool BiofilmQ. Biofilm on SS show less roughness, density, thickness and volume. The biofilm 3D structure seemed to be related to the coupon topography and roughness.The materials used in this study do not affect biofilm growth. However, their roughness and topography affect the biofilm architecture, which could influence biofilm behaviour.

Morphological changes and viability of Streptococcus mutans biofilm treated with erythrosine: A confocal laser scanning microscopy analysis.

Antimicrobial photodynamic therapy (a-PDT) is a modality that aims to induce microorganisms through visible light, a photosensitizer, and molecular oxygen. This therapy has shown promising results in controlling cariogenic biofilm in vitro and in vivo counterparts. This study investigated bacterial viability and morphological characterization of Streptococcus mutans mature biofilms after combination of erythrosine and a high potency dental curing light. Biofilms were formed on saliva-coated hydroxyapatite disks in batch culture. The samples were performed in triplicates. Fresh medium was replaced daily for five days and treated using 40 μM of E activated by HL 288 J/cm2 and total dose of 226 J at 1200 mW/cm2. Phosphate buffer saline and 0.12% of chlorhexidine were used as negative and positive control, respectively. After treatment, biofilms were assessed for microbial viability and morphological characterization by means of bio-volume and thickness. COMSTAT software was used for image analysis. Data were analyzed using two-way ANOVA followed by Tukey test with significance level 5%. The application of a-PDT and CHX treatments decreased S. mutans bacterial viability. The image analysis showed more red cells on biofilms when compared to other groups, demonstrating photobacterial killing. Erythrosine irradiated with a high potency curing light can potentially act as an antimicrobial tool in the treatment of cariogenic biofilms. The morphology and viability of microorganisms were impacted after treatment. Treatment with photodynamic therapy may be able to reduce the bio-volume and viability of bacteria present in biofilms. CLINICAL RELEVANCE AND RESEARCH HIGHLIGHTS: The use of the a-PDT technique has been applied in dentistry with satisfactory results. Some applications of this technique are in stomatology and endodontics. In the present study, we sought to understand the use of photodynamic therapy in the control of biofilm and the results found are compatible with the objective of microbiological control proposed by this technique, thus raising the alert for future studies in vivo using the combination of a-PDT with erythrosine, since they are easily accessible materials for the dental surgeon and can be applied in clinical practice.