Confocal Scanning Laser Tomography Research Articles

Pathological Role of High Sugar in Mitochondrial Respiratory Chain Defect-Augmented Mitochondrial Stress.

According to many research groups, high glucose induces the overproduction of superoxide anions, with reactive oxygen species (ROS) generally being considered the link between high glucose levels and the toxicity seen at cellular levels. Respiratory complex anomalies can lead to the production of ROS. Calcium [Ca2+] at physiological levels serves as a second messenger in many physiological functions. Accordingly, mitochondrial calcium [Ca2+]m overload leads to ROS production, which can be lethal to the mitochondria through various mechanisms. F1F0-ATPase (ATP synthase or complex V) is the enzyme responsible for catalyzing the final step of oxidative phosphorylation. This is achieved by F1F0-ATPase coupling the translocation of protons in the mitochondrial intermembrane space and shuttling them to the mitochondrial matrix for ATP synthesis to take place. Mitochondrial complex V T8993G mutation specifically blocks the translocation of protons across the intermembrane space, thereby blocking ATP synthesis and, in turn, leading to Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome. This study seeks to explore the possibility of [Ca2+]m overload mediating the pathological roles of high glucose in defective respiratory chain-mediated mitochondrial stress. NARP cybrids are the in vitro experimental models of cells with F1FO-ATPase defects, with these cells harboring 98% of mtDNA T8993G mutations. Their counterparts, 143B osteosarcoma cell lines, are the parental cell lines used for comparison. We observed that NARP cells mediated and enhanced the death of cells (apoptosis) when incubated with hydrogen peroxide (H2O2) and high glucose, as depicted using the MTT assay of cell viability. Furthermore, using fluorescence probe-coupled laser scanning confocal imaging microscopy, NARP cells were found to significantly enable mitochondrial reactive oxygen species (mROS) formation and enhance the depolarization of the mitochondrial membrane potential (ΔΨm). Elucidating the mechanisms of sugar-enhanced toxicity on the mitochondria may, in the future, help to alleviate the symptoms of patients with NARP syndromes and other neurodegenerative diseases.

Influence of Initial Root Canal Treatment on Intratubular Penetrability and Bond Strength of Nonsurgical Retreatment: An InVitro Study

IntroductionTo assess the effect of combinations of two different endodontic sealers used in initial and endodontic retreatment on the bond strength of the secondary obturation and the penetrability of the sealers. MethodsForty-eight mandibular premolars were used, receiving standardized endodontic access and biomechanical preparation. Twenty-four teeth received AH Plus sealer (AHP) in primary obturation and the others received Bio-C Sealer (BCS). Retreatment protocol was performed with an R50 instrument. The samples were further subdivided into four groups (n = 12) based on the combination of primary/secondary obturation sealers: AHP/AHP; AHP/BCS; BCS/AHP; and BCS/BCS. Four samples from each subgroup received the addition of fluorophores to the sealer for penetrability analysis using laser scanning confocal fluorescence microscopy. The root portion on the 8 push-out samples was sectioned into 6 slices of 1.0 mm. Bond strength (BS) was assessed using a universal testing machine until displacement of the filling mass. Failure pattern was evaluated under a stereomicroscope (20× magnification). BS data were analyzed using two-way analysis of variance followed by Tukey's test (P < .05), and the association between the failure pattern and BS value was assessed using the chi-square test (P < .05). Penetrability was qualitatively evaluated. ResultsThe highest BS values were observed in the AHP/AHP (4.54 ± 1.5 MPa) and BCS/AHP (5.00 ± 1.0 MPa) groups (P < .05), with a higher percentage of adhesive failures to the filling material for all groups. Laser scanning confocal fluorescence microscopy images indicated greater penetrability of AHP compared to BCS, both in initial treatment and retreatment. ConclusionAHP sealer exhibited higher BS and greater penetrability compared to BCS sealer.

Study on the Occurrence Characteristics of the Remaining Oil in Sandstone Reservoirs with Different Permeability after Polymer Flooding.

After polymer flooding, the heterogeneity between different layers intensifies, forming intricate seepage channels and fluid diversions, which results in decreased circulation efficiency and lower recovery rates, leaving a significant amount of residual oil trapped within the reservoir. Understanding the characteristics of residual oil occurrence is crucial for enhancing oil recovery post-polymer flooding. This study focused on sandstone reservoirs with varying permeability in the Saertu block of the Daqing oilfield. Using cryosectioning and laser scanning confocal microscopy, the occurrence characteristics of the residual oil in these sandstone reservoirs post-polymer flooding were investigated. Additionally, micro-CT and scanning electron microscopy were employed to analyze the impact of the pore structure on the distribution characteristics of the residual oil. The results indicate that laser scanning confocal images reveal that post-polymer flooding, the residual oil in high- and low-permeability sandstone reservoirs predominantly exists in a bound state (average > 47%), mostly as particle-adsorbed oil. In contrast, the residual oil in medium-permeability reservoirs is primarily in a free state (average > 49%), mostly as intergranular-adsorbed oil. In high-permeability sandstone reservoirs, heavy oil components are mainly in a particle-adsorbed form; in medium-permeability sandstone reservoirs, residual oil predominantly consists of heavy components, with most light components occurring in a clustered form; in low-permeability sandstone reservoirs, clustered residual oil exists in a balanced coexistence of light and heavy components, while the heavy components primarily exist in a particle-adsorbed form. Post-polymer flooding, the large pore-throat structure in high-permeability sandstone reservoirs results in effective displacement and less free residual oil; medium-permeability sandstone reservoirs, with medium-large pores and throats, have preferential channels and fine particles blocking the throats, leading to some unswept pores and more free residual oil; low-permeability sandstone reservoirs, with small pores and throats, exhibit weak displacement forces and poor mobility, resulting in more bound residual oil. The distribution and content of clay particles and clay minerals, along with the complex microscopic pore structure, are the main factors causing the differences in the residual oil occurrence states in sandstones with varying permeability.

Seasonal to interannual variations of daily growth rate of a Tridacna shell from Palau Island, western Pacific, and paleoclimatic implications

Seasonal-interannual climate change is an important component of Earth's climate system and has a significant impact on ecosystems and social systems. However, the short-run of modern observational data limits our understanding of the seasonal-interannual climates on long-term timescales. The natural archives can provide information on climate change before the industrial period, but most of their temporal resolution is too low to capture the seasonal-interannual climate signals. Tridacna spp. is the largest marine bivalve, and its shell has the potential to trace climatic/environmental changes on a daily to interannual scale. In this study, we investigated the daily growth pattern of a modern Tridacna gigas shell (PL-1) from Palau based on laser scanning confocal microscopy images. The results showed that the growth pattern of PL-1 was affected by effective solar radiation primarily, followed by nutrients on the seasonal timescale. On the interannual timescale, the growth rate of the shell was modulated by ENSO, which affected the effective solar radiation and nutrients accepted by Tridacna. The growth rate of Tridacna PL-1 was accelerated during the ENSO positive phase, and vice versa. However, due to a lack of situ observations, the effects of nutrients on Tridacna and the symbiotic zooxanthella in detail need to be further investigated. This study indicates that the daily growth rate of Tridacna shells in equatorial regions may have the potential to reconstruct seasonal-interannual climate/environment variations.

Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain

BackgroundThe channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood.MethodsWild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice.ResultsGlobal or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG.ConclusionsThe present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

Preparation, structure and luminescence properties of Ce3+: Y3(Al,Ga)5O12 translucent glass ceramics for AC-LEDs

Translucent glass ceramics containing Ce3+:Y3(Al,Ga)5O12 microcrystals were prepared by spontaneous crystallization in the SiO2–B2O3–Al2O3–Ga2O3–Y2O3–PbO–CeF3 glass system. The sample exhibits around 45 % transmittance in the range of 500–800 nm. X-ray diffraction patterns confirm the garnet phases. Scanning electron microscope and laser scanning confocal microscopy images suggest that about 5 μm grains are dispersed uniformly in the glass matrix. With increasing Ga/Al ratio in the components, both excitation and emission peaks blue shift. The emission intensity reaches to the maximum at Ga/Al ratio of 8/22, and the optimized Ce3+ content is 0.4 mol%. The sample exhibits yellowish-green persistent luminescence with lifetime of 47.3 ms. The glass ceramic-based device exhibits near standard white light emission with luminous efficiency of 64.7 lm/W, correlated color temperature of 6979 K, color rendering index of 65.4 and flicker index of 76.9 %. It indicates that the Ce3+:Y3(Al,Ga)5O12 glass ceramics are promising in the alternating current directly driven white light-emitting diodes (AC-LEDs).

Design of a Scanning Module in a Confocal Microscopic Imaging System for Live-Cell Imaging

This study proposes a Nipkow-based pinhole disk laser scanning confocal microscopic imaging system for ordinary optical microscopy, fluorescence microscopy, and confocal microscopy imaging of biological samples in order to realize the dynamic experimental monitoring of space-based life science experiments and the fine observation of biological samples. Confocal microscopic imaging is mainly completed by a scanning module that is composed of a spinning disk and other components. The parameters of the spinning disk directly determine the quality of the image. During the design process, the resolution and signal-to-noise ratios caused by different pinhole diameters in the spinning disk are the main considerations. Changes and image blurring caused by crosstalk due to the pinhole arrangement and different pinhole spacings are addressed. The high photon efficiency of the new EMCCD (electron-multiplying charge-coupled device) and CMOS (complementary metal-oxide-semiconductor) camera reduces the exposure time as much as possible, reduces damage to living cells, and achieves high-speed confocal imaging. It is shown in a confocal imaging experiment with a variable magnification of 1–40× that the imaging resolution of the system can reach a maximum of 2592 × 1944, the spatial resolution can reach 1 μm, and the highest sampling frequency is 10 fps, thus meeting the design requirements for high-speed live-cell imaging.

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.

OPTIMIZING WOUNDED ARTICULAR CARTILAGE FOR IMPROVED CARTILAGE INTEGRATION: EVALUATING CHONDROPROTECTIVE MEASURES FOR MINIMIZING IN SITU CHONDROCYTE DEATH

AbstractObjectiveThe preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective.MethodsEx vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury.ResultsIn 340mOsm media, the PCD around the 3mm cartilage wells was significantly less compared to the corresponding explants (20.05±10.24% vs 35.25±4.86%; P=0.0003). When using the 5mm biopsy punch, the PCD at the wound edges was significantly lower when compared to the 3mm cartilage wells (13.33±7.80% vs 20.05±10.24%; P=0.0121) at the same osmolarity. The width of the cell death zone for the well edges for both 3 and 5mm punches was significantly narrower when compared to their corresponding harvested cartilage explants in 340mOsm media (P&lt;0.0001; P=0.0218, respectively). Exposing cartilage to raised osmolarity (600mOsm) prior to injury significantly reduced the PCD for cartilage wells produced by the 3mm biopsy punches (from 20.05±10.24% to 12.24±6.00%; P=0.0025). In addition, the zone of cell death was marginally reduced at the edges of the 5mm cartilage wells (19.25±15.78mm to 12.72±9.09mm; P=0.0499).ConclusionsThe choice of biopsy punch and the osmolarity of the incubation medium prior to cartilage injury markedly affected the extent of chondrocyte death both at the edges of the cartilage wells and the explants. The smaller biopsy punch caused more chondrocyte death in the native cartilage wells compared to the larger punch, but this could be compensated for by the chondroprotective effect of raising the osmotic pressure. In general, there was less cell death at the wounded edges of the cartilage wells, compared to the explants. These results suggest that there is scope for further optimising the cutting implements used to create the cartilage wells and protecting chondrocytes by hyper-osmolarity in order to minimize cell death at cut edges and potentially enhance integration between cartilage repair material and host cartilage.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Quantification of lipid droplets in hatchery reared veliger larvae of the green-lipped mussel, Perna canaliculus

Domestication efforts and selective breeding in bivalve aquaculture would greatly benefit from reliable screening biomarkers in the early life stages of development (e.g., larvae). In this study the lipids remaining at the completion of embryo development in selectively-bred/hatchery reared green-lipped mussel, Perna canaliculus, were targeted and quantified using two approaches: confocal laser scanning microscopy and image analysis (CLSM/IA) on Oil Red-O stained larva (i.e., lipid droplet number and volume distribution), and Iatroscan thin layer chromatography/flame ionization detection (TLC/FID) (i.e., energetic and structural lipid classes content). Four groups of newly formed pre-feeding veliger larvae, produced from two genetically distinct families (FA, FB) that completed embryo development at either low (L, 17 °C) or high (H, 21 °C) temperatures, were evaluated. The FB produced offspring better equipped to deal with higher temperatures during embryonic development, with greater survival, higher normal development, and larger larvae than FA when developing at 21 °C. In terms of lipids content, CLSM/IA showed that each individual larva had overall between 83 and 157 lipid droplets (ranging in volume from 2.5 to 294 μm3). The TLC/FID estimation of mean total lipid content ranged from 4.1 to 6.9 ƞg larva−1 (29–39% structural lipid and 62–69% energetic lipid). Both lipid quantification methods were successful in detecting differences among larval groups with different genetic backgrounds, as well as providing insight into the ability to cope with changes in the growing environment when relying on maternal energy sources (triacylglycerol-TAG identified as the main source of energy for early development). Stronger differences between the two families were identified using CLSM/IA than with TLC/FID (i.e., with FA having overall more and larger lipid droplets). However, TLC/FID was able to distinguish FA larvae reared at high temperatures from the other groups (due to lower levels of TAG, 46% of total lipids, versus 52–56% in the other groups), therefore being more sensitive to the pooled population response compared to the individual lipid droplet quantification in CLSM/IA. The application of these tools, when fully developed, could be extended to controlled commercial operations to diagnose and predict larval performance, enabling more directed early screening and informing production and breeding decisions.

Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells.

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.

Multimodal Imaging of Dental Pulp Healing Patterns Following Tooth Autotransplantation and Regenerative Endodontic Treatment

IntroductionUnderstanding the healing process of dental pulp after tooth autotransplantation (TAT) and regenerative endodontic treatment (RET) of immature teeth is important both clinically and scientifically. This study aimed to characterize the pattern of dental pulp healing in human teeth that underwent TAT and RET using state-of-the-art imaging techniques. Materials and MethodsThis study examined 4 human teeth, 2 premolars that underwent TAT, and 2 central incisors that received RET. The premolars were extracted after 1 year (case 1) and 2 years (case 2) due to ankylosis, while the central incisors were extracted after 3 years (cases 3 and 4) for orthodontic reasons. Nanofocus x-ray computed tomography was used to image the samples before being processed for histological and immunohistochemical analysis. Laser scanning confocal second harmonic generation imaging (SHG) was used to examine the patterns of collagen deposition. A maturity-matched premolar was included as a negative control for the histological and SHG analysis. ResultsAnalysis of the 4 cases revealed different patterns of dental pulp healing. Similarities were observed in the progressive obliteration of the root canal space. However, a striking loss of typical pulpal architecture was observed in the TAT cases, while a pulp-like tissue was observed in one of the RET cases. Odontoblast-like cells were observed in cases 1 and 3. ConclusionsThis study provided insights into the patterns of dental pulp healing after TAT and RET. The SHG imaging sheds light on the patterns of collagen deposition during reparative dentin formation.

Ganoderenic acid D-loaded functionalized graphene oxide-based carrier for active targeting therapy of cervical carcinoma

Ganoderenic acid D (GAD), extracted from the Chinese herb Ganoderma lucidum, was loaded onto a graphene oxide-polyethylene glycol-anti-epidermal growth factor receptor (GO-PEG-EGFR) carrier to develop a targeting antitumor nanocomposite (GO-PEG@GAD). The carrier was fabricated from PEG and anti-EGFR aptamer modified GO. Targeting was mediated by the grafted anti-EGFR aptamer, which targets the membrane of HeLa cells. Physicochemical properties were characterized by transmission electron microscopy, dynamic light scattering, X-ray powder diffraction, and Fourier transform infrared spectroscopy. High loading content (77.3 % ± 1.08 %) and encapsulation efficiency (89.1 % ± 2.11 %) were achieved. Drug release continued for approximately 100 h. The targeting effect both in vitro and in vivo was confirmed by confocal laser scanning microscopy (CLSM) and imaging analysis system. The mass of the subcutaneous implanted tumor was significantly decreased by 27.27 ± 1.23 % after treatment with GO-PEG@GAD compared with the negative control group. Moreover, the in vivo anti-cervical carcinoma activity of this medicine was due to activation of the intrinsic mitochondrial pathway.

Effects of liquid surface tension on gas capillaries and capillary forces at superamphiphobic surfaces

The formation of a bridging gas capillary between superhydrophobic surfaces in water gives rise to strongly attractive interactions ranging up to several micrometers on separation. However, most liquids used in materials research are oil-based or contain surfactants. Superamphiphobic surfaces repel both water and low-surface-tension liquids. To control the interactions between a superamphiphobic surface and a particle, it needs to be resolved whether and how gas capillaries form in non-polar and low-surface-tension liquids. Such insight will aid advanced functional materials development. Here, we combine laser scanning confocal imaging and colloidal probe atomic force microscopy to elucidate the interaction between a superamphiphobic surface and a hydrophobic microparticle in three liquids with different surface tensions: water (73 mN m−1), ethylene glycol (48 mN m−1) and hexadecane (27 mN m−1). We show that bridging gas capillaries are formed in all three liquids. Force-distance curves between the superamphiphobic surface and the particle reveal strong attractive interactions, where the range and magnitude decrease with liquid surface tension. Comparison of free energy calculations based on the capillary menisci shapes and the force measurements suggest that under our dynamic measurements the gas pressure in the capillary is slightly below ambient.

Baking using oleogels

Baking using oleogels

Nanoparticle Coatings on Glass Surfaces to Prevent Pseudomonas fluorescens AR 11 Biofilm Formation.

Microbial colonization of surfaces is a sanitary and industrial issue for many applications, leading to product contamination and human infections. When microorganisms closely interact with a surface, they start to produce an exo-polysaccaridic matrix to adhere to and protect themselves from adverse environmental conditions. This type of structure is called a biofilm. The aim of our work is to investigate novel technologies able to prevent biofilm formation by surface coatings. We coated glass surfaces with melanin-ZnO2, melanin-TiO2, and TiO2 hybrid nanoparticles. The functionalization was performed using cold plasma to activate glass-substrate-coated surfaces, that were characterized by performing water and soybean oil wetting tests. A quantitative characterization of the antibiofilm properties was done using Pseudomonas fluorescens AR 11 as a model organism. Biofilm morphologies were observed using confocal laser scanning microscopy and image analysis techniques were used to obtain quantitative morphological parameters. The results highlight the efficacy of the proposed surface coating to prevent biofilm formation. Melanin-TiO2 proved to be the most efficient among the particles investigated. Our results can be a valuable support for future implementation of the technique proposed here in an extended range of applications that may include further testing on other strains and other support materials.

Pathological role of high sugar in mitochondrial respiratory chain defect - augmented mitochondrial stress.

Respiratory complex anomalies could lead to the production of ROS. Ca2+, at physiological levels serves as a second messenger in many physiological functions. However, a research group reported that mitochondrial calcium [Ca2+]m overload leads to ROS production which could be lethal to the mitochondria through various mechanisms. The F1F0-ATPase (ATP synthase or complex V) is the enzyme that is responsible for catalyzing the final step of oxidative phosphorylation. Mitochondrial complex V T8993G mutation specifically blocks the translocation of protons across the intermembrane space thereby blocking ATP synthesis leading to Neuropathy, Ataxia and Retinitis Pigmentosa Syndromes (NARP). The main aim undertaking this study is to explore the possibility of [Ca2+]m overload mediating the pathological roles of high glucose in defective respiratory chain mediated mitochondrial stress. The NARP cybrids are ordered and are established in vitro experimental models for the cells with F1FO-ATPase defects, these cells harbor 98% of mtDNA T8993G mutation. Their counterparts 143B osteosarcoma cell lines are the parental cell lines used for comparisons. We uncovered that, NARP cells mediated and enhanced the death of the cells (apoptosis) when incubated with hydrogen peroxide (H2O2) and high glucose respectively as depicted by the MTT assay of cell viability. Furthermore, using the fluorescence probe-coupled laser scanning confocal imaging microscopy, NARP cells significantly enabled mitochondrial reactive oxygen species (mROS) formation and enhanced the depolarization of the mitochondrial membrane potential (ΔΨm). Elucidating the mechanisms of sugar enhanced toxicity on mitochondria would help in the future to alleviate the symptoms of patients associated with Neuritis Ataxia Retinitis Pigmentosa (NARP) syndromes.

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels.

Nitric oxide (NO) plays an important role in the regulation of the immune, cardiovascular and nervous systems. Consequently, being able to monitor and quantify intracellular NO levels would provide a greater understanding of the implications of this molecule in the different biological processes, including, for example, in cancer. Here, we report a broadly applicable two-photon excitable fluorescent nanoprobe able to detect and potentially quantify NO levels in an extensive range of cellular environments. The nanoprobe consists of a thiolated photoinduced electron transfer-based two=photon fluorescent probe attached onto the surface of 2.4 ± 0.7 nm gold nanoparticles (DANPY-NO@AuNPs). The nanoprobe, which can be synthesised in a reproducible manner and exhibits great stability when stored at room temperature, is able to selectively detect NO in solution, with a dynamic range up to 150 μM, and at pH values of biological relevance. DANPY-NO@AuNPs were able to selectively detect endogenous NO in RAW264.7γ NO- macrophages and THP-1 human leukemic cells; and endogenous and exogenous NO in endothelial cells. The nanoprobe accumulated in the acidic organelles of the tested cell lines showing negligible toxicity. Importantly, DANPY-NO@AuNPs showed potential to quantify intracellular NO concentrations in MDA-MB-231 breast cancer cells. The biological evaluation of the nanoprobe was undertaken using confocal laser scanning (images and intracellular emission spectra) and multiphoton microscopies, and flow cytometry. Based on their excellent sensitivity and stability, and outstanding versatility, DANPY-NO@AuNPs can be applied for the spatiotemporal monitoring of in vitro and in vivo NO levels.

A large Stokes shift NIR fluorescent probe for visual monitoring of mitochondrial peroxynitrite during inflammation and ferroptosis and in an Alzheimer's disease model.

The excessive formation of peroxynitrite (ONOO-) in mitochondria has been implicated in various pathophysiological processes and diseases. However, owing to short emission wavelengths and small Stokes shifts, previously reported fluorescent probes pose significant challenges for mitochondrial ONOO- imaging in biological systems. In this study, a near-infrared (NIR) fluorescent probe, denoted as DCO-POT, is designed for the visual monitoring of mitochondrial ONOO-, displaying a remarkable Stokes shift of 170 nm. The NIR fluorophore of DCO-CHO is released by DCO-POT upon the addition of ONOO-, resulting in off-on NIR fluorescence at 670 nm. This phenomenon facilitates the high-resolution confocal laser scanning imaging of ONOO- generated in biological systems. The practical applications of DCO-POT as an efficient fluorescence imaging tool are verified in this study. DCO-POT enables the fluorometric visualization of ONOO- in organelles, cells, and organisms. In particular, ONOO- generation is analyzed during cellular and organism-level (zebrafish) inflammation during ferroptosis and in an Alzheimer's disease mouse model. The excellent visual monitoring performance of DCO-POT in vivo makes it a promising tool for exploring the pathophysiological effects of ONOO-.

Confocal Laser Scanning Imaging of Cell Junctions in Human Colon Cancer Cells.

The intestinal epithelium is formed by a single layer of cells. These cells originate from self-renewal stem cells that give rise to various lineages of cells: Paneth, transit-amplifying, and fully differentiated cells (as enteroendocrine, goblet cells, and enterocytes). Enterocytes, also known as absorptive epithelial cells, are the most abundant cell type in the gut. Enterocytes have the potential to polarize as well as form tight junctions with neighbor cells which altogether serve to ensure both the absorption of "good" substances into the body and the blockage of "bad" substances, among other functions. Culture cell models such as the Caco-2 cell line have been proved to be valuable tools to study the fascinating functions of the intestine. In this chapter we outline some experimental procedures to grow, differentiate, and stain intestinal Caco-2 cells, as well as image them using two modes of confocal laser scanning microscopy.