Confocal Laser Scanning Microscopy Data Research Articles

Investigating Flow-Induced Corrosion of Magnesium in Ophthalmological Milieu.

Although the impact of local fluid dynamics in the biodegradation of magnesium is well known, currently no studies in the literature address the degradation effects of ocular vitreous on bioresorbable devices made of magnesium, which could be developed as drug delivery carriers. The aim of this study was to investigate the flow-induced corrosion mechanism of magnesium in an ophthalmological environment for future applications in ophthalmic drug delivery. To achieve this, experimental and computational methods were combined. Specifically, a CFD model was employed to design experimental conditions that replicate the ocular flow-induced shear stress (FISS) on manufactured magnesium samples. Pure Mg samples were tested in a bioreactor system capable of imposing the ocular CFD calculated values of FISS on the Mg samples' surface by varying the pump flow rate. Optimal flow rates for a range of different FISS values specific to the ophthalmological fluid dynamics affecting the device were indeed determined before running the experiments. After conducting customized corrosion tests, morphological observations and profilometric maps of the eroded surfaces of Mg samples were obtained using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). These maps were then post-processed for the parametric evaluation of corrosion rates. Pre-existing localized superficial defects did affect the final corrosion pattern. SEM images and CLSM data confirmed a uniform corrosion mechanism, with corrosion rates of 1.9, 2.7, and 3.4 μm/day under different shear stress conditions (0, 0.01, and 0.032 Pa, respectively). More generally, uniform corrosion on pure Mg samples increased with higher FISS values, and at higher shear stress values (FISS = 0.032 Pa), a notable washing-out effect of the corrosion products was observed. The removal of corrosion products at higher shear stresses suggests that the dynamic ocular environment, influenced by saccadic movements, plays a significant role in the corrosion mechanism of pure magnesium. The corrosion rates determined in this study, in conjunction with clinical drug release requirements, are crucial for designing potential drug-release devices for ocular applications.

Postmodification with Polycations Enhances Key Properties of Alginate-Based Multicomponent Microcapsules.

Postmodification of alginate-based microspheres with polyelectrolytes (PEs) is commonly used in the cell encapsulation field to control microsphere stability and permeability. However, little is known about how different applied PEs shape the microsphere morphology and properties, particularly in vivo. Here, we addressed this question using model multicomponent alginate-based microcapsules postmodified with PEs of different charge and structure. We found that the postmodification can enhance or impair the mechanical resistance and biocompatibility of microcapsules implanted into a mouse model, with polycations surprisingly providing the best results. Confocal Raman microscopy and confocal laser scanning microscopy (CLSM) analyses revealed stable interpolyelectrolyte complex layers within the parent microcapsule, hindering the access of higher molar weight PEs into the microcapsule core. All microcapsules showed negative surface zeta potential, indicating that the postmodification PEs get hidden within the microcapsule membrane, which agrees with CLSM data. Human whole blood assay revealed complex behavior of microcapsules regarding their inflammatory and coagulation potential. Importantly, most of the postmodification PEs, including polycations, were found to be benign toward the encapsulated model cells.

Revolutionizing Gram-negative bacteria detection: FLIM and multicolor imaging based selective interaction study using colistin passivated carbon dots

The rising prevalence of antibiotic-resistant Gram-negative bacteria presents a major challenge for healthcare and requires the development of new and effective detection techniques. In this study, we present a novel and simple method for the selective detection of Gram-negative bacteria using multi-emissive colistin-passivated carbon dots (m-CCD) based on Fluorescence Lifetime (FLT) response and bioimaging. The m-CCD was synthesized using a one-step microwave-assisted hydrothermal process, and its interaction with Gram-negative bacteria was studied using Fluorescence Lifetime Imaging Microscopy (FLIM) and automated Confocal Laser Scanning Microscopy (CLSM). The investigations revealed a notable reduction in the FLT of m-CCD-treated Gram-negative bacteria (average lifetime of m-CCD, (τavg) m-CCD is 3.91 ns and m-CCD treated Gram-negative bacteria, (τavg) m-CCD-bac is 1.57 ns), demonstrating the selective interaction between them. Furthermore, the CLSM data revealed high specificity of m-CCD for Gram-negative bacteria, even in polymicrobial samples. Importantly, we also demonstrate that this method can quantify Escherichia coli (E. coli) cells with two linear ranges such as 3.40 × 105 – 9.80 × 105 CFU mL−1 and 6.90 × 107–4.14 × 108 CFU mL−1 in standard culture and real samples such as tap water and human urine with a limit of detection ∼ 3.68–4.89 × 104 CFU mL−1. This straightforward method has great potential for the rapid and selective detection of Gram-negative bacteria, paving the way for early diagnosis and treatment of bacterial infections.

Study of the self-assembly of a gemini surfactant induced by anionic QDs in aqueous solution.

The interaction of anionic quantum dots (QDs) with a gemini surfactant (1,1'-(propane-1,3-diyl-2-ol)bis(3-hexadecyl-1H-imidazol-3-ium)) bromide [C16Im-3OH-ImC16]Br2 in water has been investigated, yielding a unique class of luminescent self-assemblies. Instead of engaging with the QDs directly, the dimeric surfactant self-associates into micelles first. After adding [C16Im-3OH-ImC16]Br2 to aqueous solutions containing QDs, two types of structural formations, supramolecular and vesicles, were confirmed. A variety of intermediary structures, including cylindrical and oligomers of vesicles are found to be present. Field-emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM) were used to investigate the luminescent and morphological properties of the self-assembled nanostructures in the first turbid (Ti) and second turbid (Tf) regions. FESEM images show discrete spherical vesicles in the mixture's Ti and Tf regions. The presence of self-assembled QDs in these spherical vesicles makes them naturally luminescent, according to CLSM data. Because the QDs are equally distributed in the micelles, their self-quenching is greatly reduced and their luminescence is effectively sustained. We have also demonstrated the successful encapsulation of dye rhodamine B (RhB) into these self-assembled vesicles using CLSM without any structural disturbance. The discovery of luminescent self-assembled vesicles from a QD-[C16Im-3OH-ImC16]Br2 combination might lead to new potential in controlled release drug delivery and sensing.

A structure metric for quantitative assessment of fracture surfaces in 3D conceived based on confocal laser scanning microscopy data

Assessment of fracture surfaces typically involves the identification of qualitative features such as dimples, grain facets, and river patterns to determine type of fracture. The present study formulates a measure termed Real-to-Projected Area (RPA) ratio/metric to quantitatively assess fracture surfaces. Fractures surfaces of several metallic alloys tested monotonically and in fatigue to fracture including three Mg alloys, a Ti alloy in two conditions, and a Ni-based superalloy were observed using confocal laser scanning microscopy in 3D and quantitatively assessed using the metric. The metric successfully separated fracture surfaces obtained after fatigue testing from those obtained after monotonic tension testing. Specifically, the values of the metric were found to be in the range of 40–80 pct for fatigue fracture, while 90–140 pct for monotonic tension fracture. As a result, the metric can be used to distinguish between fracture in fatigue and fracture in monotonic tension when fracture types / loading modes are unknown. Moreover, the metric is sensitive to ductility of tested alloys as well as any presence of critical defects in the microstructure responsible for rapid fracture such as intermetallic inclusions. An added utility of the metric is also to identify outliers in the data such as invalid mechanical tests. • Real-to-Projected Area (RPA) ratio/metric is defined to quantitatively assess fracture surfaces. • Fracture surfaces of several alloys are observed using confocal laser scanning microscopy and quantified using the metric. • The metric can successfully separate fatigue fracture surfaces from monotonic tension fracture surfaces. • The metric is sensitive to ductility of tested alloys and defects in the microstructure such as intermetallic inclusions. • The metric can identify outliers in testing to fracture data such as invalid mechanical tests.

Surface topography data fusion of additive manufacturing based on confocal and focus variation microscopy.

In this paper, two innovative data fusion methods are proposed for reconstructing the surfaces produced by directed energy deposition (DED) additive manufacturing. The surface topographic data were obtained from confocal laser scanning microscopy (CLSM) and focus variation microscopy (FV). The first method (competitive data fusion) aims to improve the data quality by combining both the advantages of the CLSM and FV techniques, while the second method (cooperative data integration) is designed for generating a single representation that contains not only global information but also local details. The results show that both fusion methods achieved satisfactory results: in the competitive fusion, the fused data preserved the characteristics of FV data while its vertical resolution is also improved by integrating the short waves from the CLSM data; the cooperative data fusion achieved one pixel precision of the surface registration which adopted the feature-based registration method with the help of color image information. The computational complexity is reduced from O((m×n)2) to O(m×n + k). Both proposed data fusion methods provided innovative solutions for the microscopic surface reconstruction and surface representation in multiscales in the field of additive manufacturing.

Adaptive Under-Sampling Deep Neural Network for Rapid and Reliable Image Recovery in Confocal Laser Scanning Microscope Measurements

Confocal laser scanning microscopy (CLSM) is a non-destructive optical measurement system of high precision, applicable to the construction of three-dimensional (3-D) topographies of biological cells and engineered materials at the micro- and sub-micro scales. Compressive sensing (CS) has recently been applied in microscope systems to reduce the amount of sampled data required for the reconstruction of images; however, the iterative nature of the CS recovery algorithm imposes high computational complexity. This article presents an end-to-end non-iterative deep residual convolutional neural network (CNN) applicable to CLSM systems for CS-based reconstruction. In experiments and numerical simulations, the proposed scheme outperformed the existing CS recovery algorithms in terms of reconstructed image quality and computation time. The proposed algorithm also enabled the reconstruction of images using samples obtained in different regions of an image at various sampling rates to overcome nonuniform information density. The reconstruction performance of the model in terms of robustness and efficiency was validated using real-world CLSM data obtained via random scanning patterns.

Environmental conditions affect the food quality of plastic associated biofilms for the benthic grazer Physa fontinalis

With an ever-increasing amount of plastic pollution in the various aquatic ecosystems around the world, the effects on organisms are still not fully understood. Most studies focus on direct effects posed by plastic intake or entanglement, but plastic debris can also affect primary production of biofilms and have an indirect impact on its consumers. This study investigates the primary production on three common plastic types in freshwater and its food quality for a benthic grazer. We hypothesized that different polymer types affect biofilm composition as well as the life parameters of its consumers. We incubated polyethylene (PE), polyethylene terephthalate (PET) and polystyrene (PS) as well as glass (control) in a productive freshwater creek for natural biofilm establishment. To account for changes in the environmental conditions, the experiment was conducted twice during winter and late spring, respectively. These biofilms were offered to the freshwater gastropod Physa fontinalis as sole food source. Growth and reproduction of the snails were measured to monitor sublethal effects. Additionally, biofilm composition was observed using confocal laser scanning microscopy (CLSM). In winter, snails feeding off PET and PE showed a significantly lower egg production and lower growth rates were observed on PET. No such effects occurred in spring. CLSM data revealed, that algal growth was significantly lower on PE and PET during the winter treatment compared to PS and glass. Since we could only find these effects during the colder and darker months (January–March), the microbial colonization on PE and PET was inhibited by the substrate under less favorable conditions of temperature and light. Hence, benign conditions may mask the adverse effects of microplastic on food webs. Our findings show that future studies on the plastisphere will need to consider such variations to further understand the influence of plastic pollution on primary production and higher trophic levels.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis

BackgroundHyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA–EV), but their concentration and function in joint pathologies remain unknown.MethodsThe aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA–EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA–EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal–Wallis analysis of variance.ResultsThe size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups.ConclusionsCLSM analysis offers a useful tool to assess HA–EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Effects of sodium citrate on the structure and microbial community composition of an early-stage multispecies biofilm model

In recent years, most biofilm studies have focused on fundamental investigations using multispecies biofilm models developed preferentially in simulated naturally occurring low-nutrient medium than in artificial nutrient-rich medium. Because biofilm development under low-nutrient growth media is slow, natural media are often supplemented with an additional carbon source to increase the rate of biofilm formation. However, there are knowledge gaps in interpreting the effects of such supplementation on the resulting biofilm in terms of structure and microbial community composition. We investigated the effects of supplementation of a simulated freshwater medium with sodium citrate on the resulting structure, bacterial community composition, and microbial network interactions of an early-stage multispecies biofilm model. Qualitative and quantitative analyses of acquired confocal laser scanning microscopy data confirmed that sodium citrate supplementation distinctly increased biofilm biomass. Sequencing data revealed that the microbial community structure of biofilms grown in sodium citrate-supplemented conditions was characterized with increased relative abundance and dominance of Proteobacteria compared with that of biofilms grown in sodium citrate-free conditions. Our findings suggest that the supplementation of a low-nutrient medium with a carbon source in experiments involving multispecies biofilms may lead to structural and compositional biases of the microbial community, causing changes in biofilm phenotype.

The Protective Effect of Staphylococcus epidermidis Biofilm Matrix against Phage Predation.

Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms’ high tolerance to antibiotics, virulent bacteriophages were previously tested as novel therapeutic agents. However, several staphylococcal bacteriophages were shown to be inefficient against biofilms. In this study, the previously characterized S. epidermidis-specific Sepunavirus phiIBB-SEP1 (SEP1), which has a broad spectrum and high activity against planktonic cells, was evaluated concerning its efficacy against S. epidermidis biofilms. The in vitro biofilm killing assays demonstrated a reduced activity of the phage. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct planktonic and biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse planktonic cells in different physiological states, suggesting that the inefficacy for biofilm control resulted from the biofilm 3D structure and the protective effect of the matrix. To assess the impact of the biofilm architecture on phage predation, SEP1 was tested in disrupted biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells after 6 h of infection. The interaction between SEP1 and the biofilm matrix was further assessed by the addition of matrix to phage particles. Results showed that the matrix did not inactivate phages nor affected phage adsorption. Moreover, confocal laser scanning microscopy data demonstrated that phage infected cells were less predominant in the biofilm regions where the matrix was more abundant. Our results provide compelling evidence indicating that the biofilm matrix can work as a barrier, allowing the bacteria to be hindered from phage infection.

Experimental Assessment of the Performance of Two Marine Coatings to Curb Biofilm Formation of Microfoulers

Biofilms formed on submerged marine surfaces play a critical role in the fouling process, causing increased fuel consumption, corrosion, and high maintenance costs. Thus, marine biofouling is a major issue and motivates the development of antifouling coatings. In this study, the performance of two commercial marine coatings, a foul-release silicone-based paint (SilRef) and an epoxy resin (EpoRef), was evaluated regarding their abilities to prevent biofilm formation by Cyanobium sp. and Pseudoalteromonas tunicata (common microfoulers). Biofilms were developed under defined hydrodynamic conditions to simulate marine settings, and the number of biofilm cells, wet weight, and thickness were monitored for 7 weeks. The biofilm structure was analyzed by confocal laser scanning microscopy (CLSM) at the end-point. Results demonstrated that EpoRef surfaces were effective in inhibiting biofilm formation at initial stages (until day 28), while SilRef surfaces showed high efficacy in decreasing biofilm formation during maturation (from day 35 onwards). Wet weight and thickness analysis, as well as CLSM data, indicate that SilRef surfaces were less prone to biofilm formation than EpoRef surfaces. Furthermore, the efficacy of SilRef surfaces may be dependent on the fouling microorganism, while the performance of EpoRef was strongly influenced by a combined effect of surface and microorganism.

Microbial carrying capacity and carbon biomass of plastic marine debris.

Trillions of plastic debris fragments are floating at sea, presenting a substantial surface area for microbial colonization. Numerous cultivation-independent surveys have characterized plastic-associated microbial biofilms, however, quantitative studies addressing microbial carbon biomass are lacking. Our confocal laser scanning microscopy data show that early biofilm development on polyethylene, polypropylene, polystyrene, and glass substrates displayed variable cell size, abundance, and carbon biomass, whereas these parameters stabilized in mature biofilms. Unexpectedly, plastic substrates presented lower volume proportions of photosynthetic cells after 8 weeks, compared to glass. Early biofilms displayed the highest proportions of diatoms, which could influence the vertical transport of plastic debris. In total, conservative estimates suggest 2.1 × 1021 to 3.4 × 1021 cells, corresponding to about 1% of the microbial cells in the ocean surface microlayer (1.5 × 103 to 1.1 × 104 tons of carbon biomass), inhabit plastic debris globally. As an unnatural addition to sea surface waters, the large quantity of cells and biomass carried by plastic debris has the potential to impact biodiversity, autochthonous ecological functions, and biogeochemical cycles within the ocean.

Chemical efficacy of several NaOCl concentrations on biofilms of different architecture: new insights on NaOCl working mechanisms.

AimTo investigate the anti‐biofilm efficacy and working mechanism of several NaOCl concentrations on dual‐species biofilms of different architecture as well as the changes induced on the architecture of the remaining biofilms.Methodology Streptococcus oralis J22 and Actinomyces naeslundii T14V‐J1 were co‐cultured under different growth conditions on saliva‐coated hydroxyapatite discs. A constant‐depth film fermenter (CDFF) was used to grow steady‐state, four‐day mature biofilms (dense architecture). Biofilms were grown under static conditions for 4 days within a confined space (less dense architecture). Twenty microlitres of buffer, 2‐, 5‐, and 10% NaOCl were applied statically on the biofilms for 60 s. Biofilm disruption and dissolution, as well as bubble formation, were evaluated with optical coherence tomography (OCT). The viscoelastic profile of the biofilms post‐treatment was assessed with low load compression testing (LLCT). The bacteria/extracellular polysaccharide (EPS) content of the biofilms was examined through confocal laser scanning microscopy (CLSM). OCT, LLCT and CLSM data were analysed through one‐way analysis of variance (ANOVA) and Tukey’s HSD post‐hoc test. Linear regression analysis was performed to test the correlation between bubble formation and NaOCl concentration. The level of significance was set at a < 0.05.ResultsThe experimental hypothesis according to which enhanced biofilm disruption, dissolution and bubble formation were anticipated with increasing NaOCl concentration was generally confirmed in both biofilm types. Distinct differences between the two biofilm types were noted with regard to NaOCl anti‐biofilm efficiency as well as the effect that the several NaOCl concentrations had on the viscoelasticity profile and the bacteria/EPS content. Along with the bubble generation patterns observed, these led to the formulation of a concentration and biofilm structure‐dependent theory of biofilm removal.ConclusionsBiofilm architecture seems to be an additional determining factor of the penetration capacity of NaOCl, and consequently of its anti‐biofilm efficiency.

Host–pathogen interaction in Toxoplasma gondii-infected mixed chicken blood cell cultures

Toxoplasma gondii has the ability to infect various nucleated cell types in different hosts. The aim of the present study was to investigate which chicken blood cells were targeted by T. gondii in a mixed blood cell culture similar to in vivo conditions and to evaluate parasite-host cell interactions. The study consisted of two subsequent experiments. In experiment 1, we applied T. gondii tachyzoites (ME49) at a multiplicity of infection of 1tachyzoite per blood cell and examined parasite replication, cytokine, and inducible nitric oxide synthase (iNOS) mRNA expression between 1h and 48h post-infection (p.i.) by quantitative PCR. By using T. gondii RH-GFP tachyzoites expressing the green fluorescent protein (GFP) in experiment 2, we aimed for visualizing infected cells by confocal laser scanning microscopy (CLSM) and flow cytometric analysis at 24h p.i. The parasite replication curve showed a massive decrease of parasite stages until 24h p.i. followed by an approximately plateau phase. We observed mainly significantly increased iNOS mRNA expression levels in T. gondii-infected culture compared to uninfected cells. Flow cytometry and CLSM data confirmed monocytes/macrophages as main target cells for T. gondii. Moreover, different lymphocytes like B cells and cytotoxic T cells seem to be targeted to a low extent. Our findings indicate that monocytes/macrophages play a key role during T. gondii infection in chicken as host cells and triggering of immune response. To the best of our knowledge, this is the first report of a mixed chicken blood cell culture experimentally infected with T. gondii.

Structure and Fluorescence Intensity Measurements in Biofilms.

Confocal laser scanning microscopy (CLSM) is one of the most relevant technologies for studying biofilms in situ. Several tools have been developed to investigate and quantify the architecture of biofilms. However, an approach to accurately quantify the intensity of a fluorescent signal over biofilm depth is still lacking. Here we present a tool developed in the ImageJ open-source software that can be used to extract both structure and fluorescence intensity from CLSM data: BIAM (Biofilm Intensity and Architecture Measurement). This is of utmost significance when studying the fundamental mechanisms of biofilm development, differentiation, and in situ gene expression or when aiming to understand the effect of external molecules on biofilm phenotypes.

Chemical biofilm removal capacity of endodontic irrigants as a function of biofilm structure: optical coherence tomography, confocal microscopy and viscoelasticity determination as integrated assessment tools.

To investigate the influence of biofilm structure on the biofilm removal capacity of endodontic irrigants and to study changes in the architecture of the remaining biofilms. Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were cocultured under different growth conditions on saliva-coated hydroxyapatite discs. A constant depth film fermenter (CDFF) was used to grow steady-state 4-day biofilms. Biofilms were grown under static conditions for 4 and 10days within a confined space. Twenty microlitres of 2% NaOCl, 2% Chlorhexidine (CHX), 17% Ethylene-diamine-tetra-acetic acid (EDTA) and buffer were applied statically on the biofilms for 60s. Biofilm removal was evaluated with optical coherence tomography (OCT). Post-treated biofilms were assessed via low load compression testing (LLCT) and Confocal laser scanning microscopy (CLSM). Optical coherence tomography data were analysed through a two-way analysis of variance (ANOVA). Low load compression testing and CLSM data were analysed through one-way ANOVA and Dunnett's post hoc test. The level of significance was set at a<0.05. The initial biofilm structure affected the biofilm removal capacity of the irrigants. NaOCl demonstrated the greatest chemical efficacy against the biofilms and was significantly more effective on the static than the CDFF biofilms (P<0.001). CHX was ineffective and caused a rearrangement of the biofilm structure. Ethylene-diamine-tetra-acetic acid exhibited a distinct removal effect only on the CDFF biofilms. Biofilm age influenced the structure of the remaining biofilms. The 4-day grown remaining biofilms had a significantly different viscoelastic pattern compared to the respective 10-day grown biofilms (P≤0.01), especially in the NaOCl-treated group. Confocal laser scanning microscopy analysis confirmed the CHX-induced biofilm structural rearrangement. Biofilm structure is an influential factor on the chemical efficacy of endodontic irrigants. Optical coherence tomography allows biofilm removal characteristics to be studied. NaOCl should remain the primary irrigant. Ethylene-diamine-tetra-acetic acid was effective against cell-rich/EPS-poor biofilms. Chlorhexidine did not remove biofilm, but rather rearranged its structure.

Correlative imaging of biological tissues with apertureless scanning near-field optical microscopy and confocal laser scanning microscopy.

Apertureless scanning near-field optical microscopy (ASNOM) has attracted considerable interest over the past years as a result of its valuable contrast mechanisms and capabilities for optical resolutions in the nanoscale range. However, at this moment the intersections between ASNOM and the realm of bioimaging are scarce, mainly due to data interpretation difficulties linked to the limited body of work performed so far in this field and hence the reduced volume of supporting information. We propose an imaging approach that holds significant potential for alleviating this issue, consisting of correlative imaging of biological specimens using a multimodal system that incorporates ASNOM and confocal laser scanning microscopy (CLSM), which allows placing near-field data into a well understood context of anatomical relevance. We demonstrate this approach on zebrafish retinal tissue. The proposed method holds important implications for the in-depth understanding of biological items through the prism of ASNOM and CLSM data complementarity.

Evaluation of Mesial Root Canals of Mandibular Molars Obturated with Gutta-Percha and Resilon Techniques

Objective:The aim of this study was to evaluate the obturation of mesial root canals of mandibular first molars performed with different filling techniques and materials (gutta-percha and resilon).Methods:Seventy-eight mesial root canals of human mandibular first molars were prepared using the K3 rotary system, and the apical preparation was set up to size 35.04. The root canals were obturated with single cone, System B, Thermafil and Real Seal 1 techniques using either gutta-percha/ThermaSeal Plus (n=13) or Resilon/Real Seal SE (n=13). Rhodamine B dye was incorporated into the sealers. Each specimen was horizontally sectioned at 2 milimeters (mm), 4 mm and 6 mm from the apex, and the samples were examined under a stereomicroscope to evaluate the presence and type of isthmuses and the percentage areas of gutta-percha/Resilon, sealer and voids. Confocal laser scanning microscopy (CLSM) was used to evaluate the sealer penetration into dentinal tubules. The Kruskal-Wallis and Dunn’s tests were used to analyse the stereomicroscope data, while the ANOVA and Tukey tests were used to analyse the CLSM data (P<0.05).Results:Thermafil and Real Seal 1 fillings showed more gutta-percha/Resilon and less sealer (P<0.05) at the 2 mm level, but the percentage of voids was similar in all groups (P>0.05). At the 4 mm level, more sealer (P<0.05) was found in the single cone groups using both materials. The System B groups exhibited better performance at the 6 mm level. The percentage of sealer penetration showed no statistically significant differences among the obturation techniques for all evaluated levels. Similar results (P>0.05) were found for both material/sealers.Conclusion:None of the materials or techniques completely filled the mesial root canals of mandibular molars, but the plasticised techniques were more efficient. The obturations using both materials and sealers were similar.

Understanding the fundamental mechanisms of biofilms development and dispersal: BIAM (Biofilm Intensity and Architecture Measurement), a new tool for studying biofilms as a function of their architecture and fluorescence intensity

Confocal laser scanning microscopy (CLSM) is one of the most relevant technologies for studying biofilms in situ. Several tools have been developed to investigate and quantify the architecture of biofilms. However, an approach to quantify correctly the evolution of intensity of a fluorescent signal as a function of the structural parameters of a biofilm is still lacking. Here we present a tool developed in the ImageJ open source software that can be used to extract both structural and fluorescence intensity from CLSM data: BIAM (Biofilm Intensity and Architecture Measurement). This is of utmost significance when studying the fundamental mechanisms of biofilm growth, differentiation and development or when aiming to understand the effect of external molecules on biofilm phenotypes. In order to provide an example of the potential of such a tool in this study we focused on biofilm dispersion. cis-2-Decenoic acid (CDA) is a molecule known to induce biofilm dispersion of multiple bacterial species. The mechanisms by which CDA induces dispersion are still poorly understood. To investigate the effects of CDA on biofilms, we used a reporter strain of Escherichia coli (E. coli) that expresses the GFPmut2 protein under control of the rrnBP1 promoter. Experiments were done in flow cells and image acquisition was made with CLSM. Analysis carried out using the new tool, BIAM, indicates that CDA affects the fluorescence intensity of the biofilm structures as well as biofilm architectures. Indeed, our results demonstrate that CDA removes more than 35% of biofilm biovolume and suggest that it results in an increase of the biofilm's mean fluorescence intensity (MFI) by more than 26% compared to the control biofilm in the absence of CDA.