Three-dimensional Confocal Laser Scanning Microscopy Research Articles

Non-destructive monitoring and prediction of fouling by organic matters and residual anionic coagulant during membrane process

Physical fouling characteristics on silicon carbide (SiC) membranes induced by various organic matter compounds vary depending on the presence of calcium ions (Ca2+). Both destructive techniques (morphological surface analysis) and non-destructive techniques (fouling properties monitoring) were used to determine the fouling mechanisms and behavior during the membrane filtration systems. Destructive analysis and a modified Hermia model were employed to assess the fouling mechanisms. Fouling behavior was also analyzed through non-destructive monitoring techniques including optical coherence tomography (OCT) and three-dimensional laser scanning confocal microscopy (3D-LSM). At concentrations of 10, 30, and 100 mg/L without Ca2+, the flux decreased by 57–95% for humic acid (HA) and anionic polyacrylamide (APAM). APAM exhibited a notable removal rate of up to 56% without Ca2+. At concentration of 10, 30, and 100 mg/L in the absence of Ca2+, the flux decreased by 6–8% for sodium alginate (SA). However, the addition of Ca2+ led to a reduction in the flux for SA by up to 91% and resulted in a removal rate of 40%. Furthermore, addition of Ca2+ led to an alteration of the fouling characteristics of HA and SA. In the case of HA, higher concentrations resulted in elevated thickness and roughness with correlation coefficients of 0.991 and 0.992, respectively. For SA, increased SA concentration led to a thicker (correlation coefficient of 0.999) but smoother surfaces (correlation coefficients of 0.502). Monitoring of these physical characteristics of the fouling layer through non-destructive analysis is crucial for effective fouling management, optimization of the system performance and extending the lifespan of the membrane. By continuously assessing the fouling layer thickness and surface roughness, we expect to be able to provide insights on the fouling behavior, identify trends, that can help scientists and engineers to make informed decisions regarding fouling control strategies in future.

Autonomic innervation, a peripheral glial cell web, and a novel S100B expressing interstitial cell type impart structural and functional complexity to the sinoatrial node.

The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a HCN4+-pacemaker cell meshwork, which merges with a network of connexin 43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy. The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B+/GFAP- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca2þ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. The autonomic plexus, peripheral glial cell web, and a novel S100Bþ/GFAP- interstitial cell type embedded within the HCN4þ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

Coral holobiont research needs spatial analyses at the microbial scale.

Coral holobiont research needs spatial analyses at the microbial scale.

Enhanced wastewater treatment by modified basalt fiber bio-carriers: Effect of etching and surface functionalization

The application of biological carriers holds a central position in wastewater treatment for biofilm-mediated degradation processes. In this study, the surface of basalt fibers (BF) was modified by combined etching and grouping (surface functionalization via attaching glutamic acid) to produce MBFEG. The etching modification enhanced the surface properties and specific surface area of BF. However, combined etching and functionalization resulted in the successful loading of amino acids on the surface of MBFEG, ultimately improving surface hydrophilicity, adsorption properties, and biophilicity of the bio-carrier. Three-dimensional confocal laser scanning microscopy confirmed that the combined modification was highly suitable in improving microbial activity and biophilicity. The microbial communities on MBFEG bio-carrier were rich and diverse indicating a stronger degree of biogeochemical cycling, indicating enrichment of nitrogen and carbon cycles. Conclusively, etching and grouping modification technology hold a promising future for BF application in biological wastewater treatment schemes.

Plant-based oral care product exhibits antibacterial effects on different stages of oral multispecies biofilm development in vitro

BackgroundExcessive biofilm formation on surfaces in the oral cavity is amongst the main reasons for severe infection development like periodontitis and peri-implantitis. Mechanical biofilm removal as well as the use of adjuvant antiseptics supports the prevention of pathogenic biofilm formation. Recently, the antibacterial effect of the oral care product REPHA-OS®, based on medicinal plant extracts and essential oils, has been demonstrated on oral pathogens grown on agar plates. In the present study, the effectiveness of the product on medical relevant oral biofilm development should be demonstrated for the first time.MethodsAn established in vitro oral multispecies biofilm, composed of Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, was used to analyze the antibacterial effect of different REPHA-OS® concentrations on planktonic bacteria, biofilm formation and mature biofilms. It was quantified using metabolic activity assays and live/dead fluorescence staining combined with three-dimensional confocal laser-scanning microscopy. Additionally, effects on species distribution inside the biofilm were assessed by means of quantitative real-time PCR.ResultsREPHA-OS® showed statistically significant antimicrobial effects on all stages of biofilm development: a minimal inhibitory concentration of 5% could be detected for both, for planktonic bacteria and for biofilm formation. Interestingly, only a slightly higher concentration of 10% was necessary to completely kill all bacteria in mature biofilms also. In contrast, an influence on the biofilm matrix or the species distribution could not be observed. The effect could be attributed to the herbal ingredients, not to the contained ethanol.ConclusionThe strong antibacterial effect of REPHA-OS® on different stages of oral biofilm development strengthens its application as an alternative adjuvant in oral care therapies.

Intracellular bacteria are common and taxonomically diverse in cultured and in hospite algal endosymbionts of coral reefs

Corals house a variety of microorganisms which they depend on for their survival, including endosymbiotic dinoflagellates (Symbiodiniaceae) and bacteria. While cnidarian–microorganism interactions are widely studied, Symbiodiniaceae–bacteria interactions are only just beginning to receive attention. Here, we describe the localization and composition of the bacterial communities associated with cultures of 11 Symbiodiniaceae strains from nine species and six genera. Three-dimensional confocal laser scanning and electron microscopy revealed bacteria are present inside the Symbiodiniaceae cells as well as closely associated with their external cell surface. Bacterial pure cultures and 16S rRNA gene metabarcoding from Symbiodiniaceae cultures highlighted distinct and highly diverse bacterial communities occur intracellularly, closely associated with the Symbiodiniaceae outer cell surface and loosely associated (i.e., in the surrounding culture media). The intracellular bacteria are highly conserved across Symbiodiniaceae species, suggesting they may be involved in Symbiodiniaceae physiology. Our findings provide unique new insights into the biology of Symbiodiniaceae.

Neuroglia infection by rabies virus after anterograde virus spread in peripheral neurons

The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS). Recent 3D imaging of field RABV-infected brains revealed a remarkably high proportion of infected astroglia, indicating that highly virulent field viruses are able to suppress astrocyte-mediated innate immune responses and virus elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population of field RABV. This suggests that virus release from axons and infected SCs is part of the RABV in vivo cycle and may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of RABV in axon-surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for successful transport from the peripheral infection sites to the brain.

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During Aspergillus fumigatus-Induced Inflammation.

People are constantly exposed to airborne fungal spores, including Aspergillus fumigatus conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense A. fumigatus conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the A. fumigatus conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII+ antigen-presenting cells, including IE-DCs, and CD11b+ phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of A. fumigatus conidia increased the number of CD11b+ phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b+ phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b+ phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b+ phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b+ phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.

Visible-Light-Induced Photocatalytic and Antibacterial Activity of TiO2 Codoped with Nitrogen and Bismuth: New Perspectives to Control Implant-Biofilm-Related Diseases.

Biofilm-associated diseases are one of the main causes of implant failure. Currently, the development of implant surface treatment goes beyond the osseointegration process and focuses on the creation of surfaces with antimicrobial action and with the possibility to be re-activated (i.e., light source activation). Titanium dioxide (TiO2), an excellent photocatalyst used for photocatalytic antibacterial applications, could be a great alternative, but its efficiency is limited to the ultraviolet (UV) range of the electromagnetic spectrum. Since UV radiation has carcinogenic potential, we created a functional TiO2 coating codoped with nitrogen and bismuth via the plasma electrolytic oxidation (PEO) of titanium to achieve an antibacterial effect under visible light with re-activation potential. A complex surface topography was demonstrated by scanning electron microscopy and three-dimensional confocal laser scanning microscopy. Additionally, PEO-treated surfaces showed greater hydrophilicity and albumin adsorption compared to control, untreated titanium. Bismuth incorporation shifted the band gap of TiO2 to the visible region and facilitated higher degradation of methyl orange (MO) in the dark, with a greater reduction in the concentration of MO after visible-light irradiation even after 72 h of aging. These results were consistent with the in vitro antibacterial effect, where samples with nitrogen and bismuth in their composition showed the greatest bacterial reduction after 24 h of dual-species biofilm formation ( Streptococcus sanguinis and Actinomyces naeslundii) in darkness with a superior effect at 30 min of visible-light irradiation. In addition, such a coating presents reusable photocatalytic potential and good biocompatibility by presenting a noncytotoxicity effect on human gingival fibroblast cells. Therefore, nitrogen and bismuth incorporation into TiO2 via PEO can be considered a promising alternative for dental implant application with antibacterial properties in darkness, with a stronger effect after visible-light application.

Effects of carbon soot from the combustion of diesel fuels on the tribological properties of lubricating oil and diesel fuels

PurposeThis paper aims to explore the effects of mineral diesel fuel carbon soot (MCS) and biodiesel carbon soot (BCS) on the lubrication of polyalphaolefin (PAO) and diesel fuels.Design/methodology/approachTwo styles of carbon soot were prepared from the natural combustion of mineral diesel fuel oil (MDO) and biodiesel oil (BDO). Tribological tests were conducted on a high-frequency reciprocating rig. Friction surfaces were characterized using three-dimensional laser scanning confocal microscopy and Raman spectroscopy.FindingsThe addition of MCS and BCS to PAO could reduce friction in most cases. MCS had a negligible effect on the wear for contents not exceeding 1.0 per cent. By contrast, BCS exhibited a considerable negative influence on the wear resistance even at low contents. For diesel fuels, MCS reduced both friction and wear, whereas BCS substantially deteriorated the lubrication of BDO. MCS formed a Fe3O4/C composite lubricating film on the friction surface. BCS also entered the contact region, but it did not form an effective Fe3O4/C composite lubricating film.Originality/valueThis work compared MDO and BDO from a different perspective, i.e. the effects of their combustion carbon soot on the lubrication of lubricating oil and fuel oil. The significant negative effect of BCS on the lubrication of lubricating oil and BDO is a problem that could occur in the industrial application of BDO.

Enhanced Corrosion Resistance of Superhydrophobic Layered Double Hydroxide Films with Long-Term Stability on Al Substrate.

A superhydrophobic ZnAl-layered double hydroxide (LDH)-La film was prepared by a hydrothermal method and further modification by laurate anions in this work. Comprehensive characterizations of this film were performed in terms of morphology, composition, structure, roughness, and wettability by scanning electronic microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, three-dimensional laser scanning confocal microscopy. The long-term corrosion protection effect of this superhydrophobic film was investigated deeply by monitoring the changes of the electrochemical impedance spectra for a long time of up to a month in 3.5 wt % NaCl solution. In the meantime, the changes of the contact angle were also recorded with the evolution of the immersion time. The result indicated that the stable superhydrophobic ZnAl-LDH-La film was able to provide efficient protection for the underlying Al substrate for a long time. In addition, the capability of the superhydrophobic surface against harsh conditions, including chemical damages and physical damages, was emphatically investigated. It was found that the superhydrophobic surface was chemically stable toward acid (pH ≥ 3), alkali, and heating, and it also exhibited high ultraviolet (UV) radiation resistance. This superhydrophobic coating maintained superhydrophobicity for 7 days of radiation in an UV chamber equipped with a 40 W UV lamp (λ = 254 nm), indicating superior ability of adapting to outdoor environment. This comprehensive investigation of the superhydrophobic ZnAl-LDH-La film is considerably helpful for researchers and engineers to get deep insight into its potential for practical applications in the field of corrosion and protection.

Aspergillus fumigatus Infection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice.

Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall after Aspergillus fumigatus conidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1+ or Ly6G+ neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h after A. fumigatus infection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates upon A. fumigatus infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

A three-dimensional statistical model for imaged microstructures of porous polymer films.

A thresholded Gaussian random field model is developed for the microstructure of porous materials. Defining the random field as a solution to stochastic partial differential equation allows for flexible modelling of nonstationarities in the material and facilitates computationally efficient methods for simulation and model fitting. A Markov Chain Monte Carlo algorithm is developed and used to fit the model to three-dimensional confocal laser scanning microscopy images. The methods are applied to study a porous ethylcellulose/hydroxypropylcellulose polymer blend that is used as a coating to control drug release from pharmaceutical tablets. The aim is to investigate how mass transport through the material depends on the microstructure. We derive a number of goodness-of-fit measures based on numerically calculated diffusion through the material. These are used in combination with measures that characterize the geometry of the pore structure to assess model fit. The model is found to fit stationary parts of the material well.

Superparamagnetic iron oxide-encapsulating polymersome nanocarriers for biofilm eradication.

The rising prevalence and severity of antibiotic-resistant biofilm infections poses an alarming threat to public health worldwide. Here, biocompatible multi-compartment nanocarriers were synthesized to contain both hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) and the hydrophilic antibiotic methicillin for the treatment of medical device-associated infections. SPION co-encapsulation was found to confer unique properties, enhancing both nanocarrier relaxivity and magneticity compared to individual SPIONs. These iron oxide-encapsulating polymersomes (IOPs) penetrated 20μm thick Staphylococcus epidermidis biofilms with high efficiency following the application of an external magnetic field. Three-dimensional laser scanning confocal microscopy revealed differential bacteria death as a function of drug and SPION loading. Complete eradication of all bacteria throughout the biofilm thickness was achieved using an optimized IOP formulation containing 40μg/mL SPION and 20μg/mL of methicillin. Importantly, this formulation was selectively toxic towards methicillin-resistant biofilm cells but not towards mammalian cells. These novel iron oxide-encapsulating polymersomes demonstrate that it is possible to overcome antibiotic-resistant biofilms by controlling the positioning of nanocarriers containing two or more therapeutics.

Surface microtexturing of Ti–6Al–4V using an ultraviolet laser system

The modifications of implant surfaces are known to enhance their osseointegration potential. The objective of this study was to develop an efficient method of increasing surface roughness with a low energy pulsed ultraviolet (UV) laser. The UV laser treatment was applied on six titanium alloy (Ti–6Al–4V) disks with machined surfaces and a commercially available implant to offered a favorable surface topography for osseointegration. Three-dimensional confocal laser-scanning microscopy was used to observe the surface structures. Osteogenic cells were grown on the Ti–6Al–4V surfaces to assess their biocompatibility. The investigators found that the surface roughness obtained with the UV laser was similar to other SLA surfaces previously described. In addition, the UV laser-ablated surfaces exhibited significant texturing in surface topography and sustained osteogenic cells growth on the treated surfaces.

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.

A theoretical analysis and experimental verification of a laser drilling process for a ceramic substrate

This paper reports theoretical and experimental methods for drilling alumina ceramic substrates by using a nanosecond-pulsed ultraviolet laser. A physical model was established using ANSYS parameter design language finite element software. The influence of laser parameters, such as laser fluence, number of pulses and its duration, and frequency, on morphology—depth, diameter, taper, and temperature distribution—was investigated using three-dimensional confocal laser scanning microscopy. Simulation and experimental results reveal that higher laser fluence and number of pulses produces larger drilling depths and diameters. Laser fluence, pulse duration, frequency, and pulse number of 5.10 J/cm2, 1000 μs, 20 kHz, and >9, respectively, can successfully drill through the alumina ceramic substrate. Pulse duration between 3 and 8 ms yielded the smallest hole taper.

Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk

The objective of this study was to determine the contribution of phospholipids from buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The aggregation mechanisms were characterised using analysis with Ellman's reagent, one-dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that phospholipids from buttermilk were involved in the formation of protein aggregates through the MFGM fragments at a low temperature, whereas phospholipids could interact directly with the proteins at a higher temperature (80 °C).

The structure and component characteristics of partial nitrification biofilms under autotrophic and heterotrophic conditions

The differences in the structure and component characteristics of partial nitrification biofilms between autotrophic and heterotrophic conditions were investigated in this work. Three-dimensional excitation-emission matrix fluorescence spectroscopy (EEM), fluorescence staining, and confocal laser scanning microscopy (CLSM) were used to determine differences in the architecture and extracellular polymeric substance (EPS) distribution of the autotrophic and heterotrophic biofilms. Partial nitrification was successfully achieved, and the results demonstrated that an appropriate amount of organic carbon (chemical oxygen demand (COD)/N = 2.6) is advantageous for obtaining better partial nitrification. The final ammoniation and nitrosation rates achieved were 97 and 99 %, respectively. Proteins (PN) and polysaccharides (PS) were dominant in the tightly bound EPS (TB-EPS) of autotrophic and heterotrophic biofilms, with PN/PS ratios of 0.96 and 0.69, respectively. Proteins, lipids, α-D-glucopyranose polysaccharides, and nucleic acids were mostly present within the layers of biofilms, but they were distributed in the upper-middle portion of the autotrophic biofilm and increased with depth from the upper layer in the heterotrophic biofilms.

Permeabilization Assay for Antimicrobial Peptides Based on Pore-Spanning Lipid Membranes on Nanoporous Alumina

Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 μm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.