Direct Microscopic Observation Research Articles

Amorphous to fcc-polycrystal transition in Ge2Sb2Te5 thin films studied by electrical measurements: Data analysis and comparison with direct microscopy observations

We experimentally investigate the isothermal amorphous-to-fcc polycrystalline phase transition process in amorphous Ge2Sb2Te5 thin films prepared by sputtering. The amorphous layers were either as deposited or formed by Ar+ ion implantation after crystallization at 300 °C. The kinetics of the amorphous-to-polycrystal transition are analyzed through electrical measurements, in which the Johnson–Mehl–Avrami–Kolmogorov theory is employed. The procedure to extract the kinetics of the phase transition from conductivity versus time data is carefully discussed and compared to data of cross-sectional transmission electron microscopy images versus anneal time. By following this proposed procedure, the nucleation and growth parameters, and the activation energies have been determined. Results indicate that the process of isothermal crystallization in Ge2Sb2Te5 takes place in two stages, in which the Avrami exponent changes in the range from 3 to 1. These results are understood in terms of modifications in the kinetics of the phase transition.

Direct observation of bacterial deposition onto clean and organic-fouled polyamide membranes

Nanofiltration (NF) and reverse osmosis (RO) membranes are commonly applied to produce highly purified water from municipal wastewater effluents. In these applications, biofouling limits overall process performance and increases the cost of operation. Initial bacteria adhesion onto a membrane surface is a critical early step in the overall process of membrane biofouling. However, adsorption of effluent organic matter onto the membrane may precede bacterial deposition and change membrane surface properties. Herein we employed direct microscopic observation to elucidate mechanisms governing bacterial cell deposition onto clean and organic-fouled NF and RO membranes. Bovine serum albumin (BSA) and alginic acid (AA) were used as models for protein and polysaccharide rich organic matter in secondary wastewater effluents. In all experiments, organic fouling increased membrane hydraulic resistance and salt rejection, in addition to interfacial hydrophilicity and roughness. Even though surface hydrophilicity increased, the rougher surfaces presented by organic-fouled membranes produced nano-scale features that promoted localized bacterial deposition. An extended DLVO analysis of bacterial cells and membrane surface properties suggested that bacterial deposition correlated most strongly with the Lewis acid–base free energy of adhesion and root mean square (RMS) roughness, whereas van der Waals and electrostatic free energies were weakly correlated. This was true for both clean and organic-fouled membranes. Bacterial deposition rates were clearly influenced by an antagonistic interplay between macroscopic surface hydrophilicity and nano-scale surface roughness.

Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions

Abstractζ‐potential measurements on LUVs allow to evidence the influence of pH, ionic salt concentration, and polyelectrolyte charge on the interaction between polyelectrolyte (chitosan and hyaluronan) and zwitterionic lipid membrane. First, chitosan adsorption is studied: adsorption is independent on the chitosan molecular weight and corresponds to a maximum degree of decoration of 40% in surface coverage. From the dependence with pH and independence with MW, it is concluded that electrostatic interactions are responsible of chitosan adsorption which occurs flat on the external surface of the liposomes. The vesicles become positively charged in the presence of around two repeat units of chitosan added per lipid accessible polar head in acid medium down to pH = 7.2. Direct optical microscopy observations of GUVs shows a stabilization of the composite liposomes under different external stresses (pH and salt shocks) which confirms the strong electrostatic interaction between the chitosan and the lipid membrane. It is also demonstrated that the liposomes are stabilized by chitosan adsorption in a very wide range of pH (2.0 < pH < 12.0).Then, hyaluronan (HA), a negatively charged polyelectrolyte, is added to vesicles; the vesicles turn rapidly negatively charged in presence of adsorbed HA Finally, we demonstrated that hyaluronan adsorbs on positively charged chitosan‐decorated liposomes at pH < 7.0 leading to charge inversion in the liposome decorated by the chitosan‐hyaluronan bilayer. Our results demonstrate the adsorption of positive and/or negative polyelectrolyte at the surface of lipidic vesicles as well as their role on vesicle stabilization and charge control.

Giant unilamellar vesicles - a perfect tool to visualize phase separation and lipid rafts in model systems.

Model systems such as black lipid membranes or conventional uni- or multilamellar liposomes are commonly used to study membrane properties and structure. However, the construction and dimensions of these models excluded their direct optical microscopic observation. Since the introduction of the simple method of liposome electroformation in alternating electric field giant unilamellar vesicles (GUVs) have become an important model imitating biological membranes. Due to the average diameter of GUVs reaching up to 100 microm, they can be easily observed under a fluorescent or confocal microscope provided that the appropriate fluorescent probe was incorporated into the lipid phase during vesicle formation. GUVs can be formed from different lipid mixtures and they are stable in a wide range of physical conditions such as pH, pressure or temperature. This mini-review presents information about the methods of GUV production and their usage. Particularly, the use of GUVs in studying lipid phase separation and the appearance and behavior of lipid domains (rafts) in membranes is discussed but also other examples of GUVs use in membrane research are given. The experience of the authors in setting up the GUV-forming equipment and production of GUVs is also presented.

Onychomycosis in Tehran: mycological study of 504 patients

Onychomycosis is a common nail disorder resulting from the invasion of the nail plate by a dermatophyte, yeast or mould species and gives rise to some diverse clinical presentations. The purpose of the present study was to isolate and identify the causative fungi of onychomycosis in the population of Tehran, Iran. Nail samples from 504 patients with prediagnosis of onychomycosis during 2005 were examined both by direct microscopical observation of fungal elements in KOH preparations and in culture for the identification of the causative agent. All samples were inoculated on (i) Sabouraud dextrose agar (SDA, Merck), (ii) SDA with 5% chloramphenicol and cycloheximide in duplicate for dermatophyte and (iii) SDA with 5% chloramphenicol in triplicate for mould isolation. The criteria for the diagnosis of onychomycosis caused by non-dermatophytic moulds were based on microscopical observation of fungal elements, growth of the same mould in all triplicate culture and no growth of a dermatophyte or yeast in all the cultures. Of 504 cases examined, 216 (42.8%) were mycologically proven cases of onychomycosis (144 fingernails, 72 toenails). Among the positive results, dermatophytes were diagnosed in 46 (21.3%), yeasts in 129 (59.7%) and non-dermatophytic moulds in 41 (19%). Trichophyton mentagrophytes was the most common causative agent (n = 22), followed by Trichophyton rubrum (n = 13), Candida albicans (n = 42), Candida spp. (n = 56) and Aspergillus spp. (n = 21). Nearly half of the clinically suspected fungal nail infections are onychomycosis and yeast is responsible for most of the infections in Iran.

Energetics of Cholesterol Transfer between Lipid Bilayers

It is believed that natural biological membranes contain domains of lipid ordered phase enriched in cholesterol and sphingomyelin. Although the existence of these domains, called lipid rafts, is still not firmly established for natural membranes, direct microscopic observations and phase diagrams obtained from the study of three-component mixtures containing saturated phospholipids, unsaturated phospholipids, and cholesterol demonstrate the existence of lipid rafts in synthetic membranes. The presence of the domains or rafts in these membranes is often ascribed to the preferential interactions between cholesterol and saturated phospholipids, for example, between cholesterol and sphingomyelin. In this work, we calculate, using molecular dynamics computer simulation technique, the free energy of cholesterol transfer from the bilayer containing unsaturated phosphatidylcholine lipid molecules to the bilayer containing sphingomyelin molecules and find that the affinity of cholesterol to sphingomyelin is higher. Our calculations of the free-energy components, energy and entropy, show that cholesterol transfer is exothermic and promoted by the favorable change in the lipid-lipid interactions near cholesterol and not by the favorable energy of cholesterol-sphingomyelin interaction, as assumed previously.

Prevalence of Bacterial Vaginosis and Impact of Genital Hygiene Practices in Non-Pregnant Women in Zanjan, Iran

Bacterial vaginosis is one of the most common causes of reproductive tract infection (RTI), it's prevalence is influenced by many factors. The aim of this study is to determine the prevalence of bacterial vaginosis and impact of sexual and genital hygienie practices and socio-demographic characteristics in non pregnant women of Zanjan province in Iran. 500 non-pregnant, married women were randomly selected for this study. This is a descriptive-analytic study conducted among non-pregnant referred to primry healthcare centres in Zanjan between May to August 2006. Following gynecological examination and vaginal sample collection by physicians, bacterial vaginosis was confirmed by Nugent criteria, tricomoniasis by direct microscopy and candidiasis by direct microscopic observation and evaluation of presenting clinical signs of vulvovaginitis. The prevalence of RTI was 27.6%. Out of which 16.2% was devoted to bacterial vaginosis (BV), 6.6% to trichomoniasis and 4.8% to Vulvovaginal candidiasis (VVC). In contrast to coital hygiene, there was a significant correlation between menstrual and individual vaginal hygiene and BV (p<0.01 and p<0.001) respectively. There was a significant correlation between BV and education (p<0.025), number of pregnancy (p<0.05) and method of contraception (p<0.005). No significant correlation was observed between age, age of marriage and abortion. The data obtained suggests that the prevalence rate of BV is relatively high and could be affected by hygiene behaviors and certain socio-demographic characteristics, which indicate the need for comprehensive, scheduled programs of healthcare educations, aimed at reducing BV prevalence.

2P-174 静磁場下における人口脂質膜のダイナミクスの光学顕微鏡観察(2)(生体膜・人工膜-ダイナミクス,第47回日本生物物理学会年会)

2P-174 静磁場下における人口脂質膜のダイナミクスの光学顕微鏡観察(2)(生体膜・人工膜-ダイナミクス,第47回日本生物物理学会年会)

Biodeterioration and restoration of a 16th-century book using a combination of conventional and molecular techniques: A case study

In this paper we deliver a report on the study of microbiological damage found on the pages of a 16th-century book. Our aim is to describe the procedures needed to ensure a conservative approach to the restoration of valuable books and objects of art made from, or supported on, paper. The techniques employed to evaluate and describe the damage observed, as well as the organisms responsible for biodeterioration, are discussed. A range of sampling techniques and instruments were utilised, including swabs and adhesive tape. Conventional methods, such as classic culturing and the direct microscopic observation of sampled material, were coupled with DNA-fingerprinting and phylogenetic analysis. We postulated that the purple stains which migrate through the pages with a felted consistency (Fig. 2), based on all the information obtained using traditional and molecular means, were caused by a cellulolytic fungus producing purple essudates, characterised by echinated conidia and Hülle cells. These elements were consistent with the discovery of both A. versicolor and A. nidulans using molecular techniques.

Dye fluorescence enhancement and quenching by gold nanoparticles: Direct near-field microscopic observation of shape dependence

We investigated the enhancement and quenching of dye fluorescence by single gold nanoparticles using an aperture-type scanning near-field optical microscope to evaluate the shape dependence. Enhancement or reduction of fluorescence was found to be strongly dependent on the shape of the particle. Gold nanoplates showed significant fluorescence enhancement, as did aggregated nanoparticles for the most part. On the other hand, gold nanorods with high aspect ratios showed quenching effects. The results suggest that the electromagnetic near-field in the vicinity of the surface of nanoparticles, not just near the apex or the tip but the whole surface, is important for understanding fluorescence enhancement and quenching.

Direct observations of the kinetics of migrating T cells suggest active retention by endothelial cells with continual bidirectional migration

The kinetics and regulatory mechanisms of T cell migration through the endothelium have not been fully defined. In experimental, filter-based assays in vitro, transmigration of lymphocytes takes hours, compared with minutes, in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates, or collagen gels and treated them with TNF-α, IFN-γ, or both prior to analysis of lymphocyte migration in the presence or absence of flow. PBL, CD4+ cells, or CD8+ cells took many hours to migrate through EC-filter constructs for all cytokine treatments. However, direct microscopic observations of EC filters, which had been mounted in a flow chamber, showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After a brief settling without flow, PBL and isolated CD3+ or CD4+ cells crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes migrated back and forth continuously across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were altered little. On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours. In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration in which EC support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration.

Electrochemomechanical properties of aniline/2-methoxyaniline copolymers

Copolymers of aniline (ANI) and 2-methoxyaniline (MOA) in variable ratios, as well as the corresponding homopolymers, were electrosynthesized and characterized by elemental analysis, IR spectroscopy, and direct laser desorption ionization. The resulting copolymer composition obtained from the analysis was correlated with the feed composition through the Mayo–Lewis equation, confirming the higher reactivity of MOA. The electrochemomechanical behaviour was studied through direct microscopical observation of the volume changes caused by redox switching in sulphuric and perchloric acid media. It was found that the freshly electropolymerized films had, for the same voltammetric charge, smaller volume as the MOA/ANI ratio increased. The volume changes observed during the first cycle where consequently strongly dependent on the polymer composition, but also on the electrolyte anion. The stationary cycle changes showed less dependence on both. The behaviour reveals different anion/polymer interactions as the copolymer composition is changed. From the perspective of mechanical applications the ANI–MOA copolymers, for most of the composition range, did not show marked differences compared with poly(aniline), thus being materials with similar characteristics to this homopolymer but being more processable.

Micro- and nanomanipulation inside the SEM

Based on individual piezodriven nanomotoric units a nanomanipulation platform is integrated into a low voltage field emission SEM (Zeiss Gemnini). The instrumentation allows the controlled movement of individual particles (ca. 50 nm), the cutting of ultrathin polymer membranes or the deformation of hollow polymer spheres with an ultrathin elastic membrane (ca. 20 nm thickness). Interaction with the objects can be done under direct microscopic observation. Imaging up to 20000 x magnification without any detectable influences of external vibrations guarantees the precise manipulation. Under appropriate imaging conditions (e.g. using low energy electrons) specimen charging can be avoided and even uncoated glass tips can be used to manipulate samples.

Characterization ofSclerotiniaand mycoparasiteConiothyrium minitansinteraction by microscale co-culture

To characterize the interaction of Sclerotinia sclerotiorum and S. minor with strains of the mycoparasite and commercial biocontrol agent Coniothyrium minitans using novel perfusion chamber gasket co-culture. Sclerotinia were cultured in perfusion chamber gaskets and then flooded with Coniothyrium conidia. After germination, Coniothyrium failed to show any form of directed growth, making contact with Sclerotinia hyphae in a random manner. In turn, some Coniothyrium hyphae coiled round Sclerotinia counterparts and although no intracellular growth was observed, Coniothyrium proliferated, while the hyphae of Sclerotinia became vacuolated and lost the cytoplasm. When co-cultures of Sclerotinia with Coniothyrium were flooded with FITC-lectins, small difference in fluorescence between the fungi was found with FITC-Con A suggesting that cell walls of both the species exposed mannose. In contrast, Coniothyrium fluoresced poorly in comparison with Sclerotinia when FITC-wheat germ agglutinin was used, indicating a marked paucity of N-acetylglucosamine exposure by cell walls of Coniothyrium, hence reduced exposure to chitinolytic enzyme action. The approach employed supported direct sequential microscopic observation of Coniothyrium and Sclerotinia as well as the utilization of representative fluorescent moieties to characterize relative carbohydrate cell wall exposure.

Application of interference microscopy to the study of hologram build-up in LiNbO3 crystals

Interference microscopy was applied to direct microscopic observation of temporal evolution of phase holograms in LiNbO3:Fe photorefractive crystals. First a hologram was recorded in the sample, and diffraction efficiency was monitored during hologram build-up using inactinic laser light. Thus kinetics of hologram build-up could be determined. The initial hologram was erased using white light. Then a series of write-erase cycles were performed with increasing exposure times. Holograms were observed by interference microscope after each exposure. The time elapsed between the exposure and the microscopic observation was negligible compared to the relaxation time of the hologram. The obtained temporal evolution of the grating profile gives a deeper insight into the physical mechanism of hologram formation in photorefractive materials than simple diffraction efficiency measurements. A congruently grown sample of LiNbO3 doped with 10−3mol/mol Fe in melting was studied by this method. Sample thickness was set to 300μm to allow correct microscopic observation. Plane-wave holograms were recorded in the samples using an Ar–ion laser at λ=488.0nm of grating constants of 3, 6.5 and 8.8μm.

The Abdominal Adipofascial Flap

The peripheral adipose tissue is a vital component of many procedures in reconstructive and esthetic surgery. There is a limited knowledge on hemodynamic changes of adipose tissue. A direct intravital microscopic observation method is needed for the measurement of microcirculatory changes occurring in peripheral fat tissue during different plastic surgical procedures. Here we are introducing a model allowing for the direct in vivo monitoring and measurements of microcirculatory hemodynamics of peripheral adipose tissue. Eight male Lewis rats weighing between 150 and 180 g were used in this study. Eight abdominal adipofascial flaps based on the left femoral artery, vein, and nerve were dissected after excision of the skin of the groin and lower abdominal region. Flap angiography using Indian ink was performed to demonstrate vascular anatomy of the flap. Standard intravital microscopy was used to monitor hemodynamic parameters such as vascular diameters, functional capillary perfusion, and leukocyte-endothelial interactions. Under direct intravital microscopy, microcirculatory parameters including vascular diameters, capillary perfusion, and leukocyte-endothelial interaction behaviors of the abdominal adipofascial flaps were established. We have showed feasibility of monitoring microcirculatory hemodynamics of the abdominal adipofascial flap model in rat. This model can be applied for intravital recordings of peripheral adipose tissue physiology and in different research scenarios such as the effects of ischemia reperfusion injury, effects of surgical trauma and wound healing studies with application of different pharmacologic agents and treatment protocols.

Differences in viability and reactivity of actinospores of three myxozoan species upon ageing

Little is known about the viability of myxozoan actinospore stages after harvest from laboratory cultures of infected oligochaete worms. The viability and reactivity of actinospores of three myxozoan species was evaluated after short-term storage at 4 degrees C and 12 degrees C. Two methods of determining actinospore viability were compared: differential fluorescent staining and direct microscopic observation of morphological indicators of spore integrity. Spore reactivity was quantified by measuring polar filament discharge rates in a micro-assay with fish mucus substrate and mechanical stimulation by vibration. The age-dependent viability of the three species showed clear differences. Myxobolus cerebralis actinospores had the shortest effective life span whereas Henneguya nuesslini actinospores survived significantly longer. Storage at lower temperatures yielded higher viability in all species. Myxobolus pseudodispar actinospores were significantly robust up to 12 degrees C when assessed by staining, but showed similar viability characteristics as H. nuesslini when analyzed morphologically. Evaluation of spore viability by fluorescent staining correlated with morphological assessment, although fewer viable actinospores were usually detected microscopically. Polar filament discharge activity of morphologically intact actinospores did not significantly decrease until the third day of storage compared to freshly harvested samples. The results indicate that durability and reactivity trends during storage of actinospores differ among myxozoan species.

Direct observation of initial microbial deposition onto reverse osmosis and nanofiltration membranes

Physicochemical factors governing initial rates of microbial deposition onto reverse osmosis (RO) and nanofiltration (NF) membranes were studied using direct microscopic observation. Deposition rates for three different microorganisms onto four NF/RO membranes increased with (a) increasing permeate velocity, (b) decreasing crossflow velocity, (c) increasing salt rejection, (d) increasing cell size, (e) increasing membrane surface roughness, (f) decreasing (cell-membrane) free energy of adhesion, and (g) decreasing (cell-membrane) electrostatic double layer repulsion. Relatively hydrophilic, smooth surfaces produced macroscopically repulsive interfacial interactions (i.e., cells should not deposit); however, microscopic heterogeneities – which appear inherent to interfacially polymerized polyamide thin films – facilitated sufficient microbial deposition onto clean membranes that microcolonies formed within minutes to hours. At fluxes greater than ∼8 μm s −1 (∼20 lmh or 16 gfd), permeate drag forces overwhelmed repulsive interfacial forces leading to purely convective deposition. Concentration polarization of rejected salt ions destabilized cells and enhanced deposition onto NF/RO membrane surfaces. The complex interplay between membrane surface properties, concentration polarization, and hydrodynamics was reasonably captured by a simple, analytical interfacial force model. These results help explain why polyamide NF/RO membranes are prone to bacterial adhesion and how the balance between crossflow and permeate hydrodynamics governs initial microbial deposition onto clean NF/RO membranes.

Role of hydrophobic natural organic matter flocs on the fouling in coagulation-membrane processes

Effects of the properties of flocs consisting of natural organic matter (NOM) contained in surface waters on membrane fouling in the coagulation-microfiltration (MF) treatment were examined in this study. Two types of waters containing different quantities of dissolved NOM primarily composed of humic substances were employed, low humic natural water and high humic lab-prepared water. Physical properties of the flocs formed in the coagulation process were analyzed by flocculation index (FI), fractal dimension, and microscopic observations. Flocs of high humic water were small and regular in shape, represented by the low FI and high fractal dimension, indicating that these flocs can be more easily compressible than those of low humic natural water. This was confirmed by direct microscopic observation and increased compressibility of the cakes formed with the high humic water flocs on the membrane surface. As a result, membrane fouling became pronounced, supported by increase of specific cake resistance and substantial decrease of water flux through the membrane. It is our expectation that these results provide useful information for the control of membrane fouling caused by flocs in the coagulation-MF membrane treatment.

Evaporative Self-Assembly from Complex DNA−Colloid Suspensions

Evaporation-induced pattern formation has attracted considerable attention as a simple yet versatile method for generating self-assembled structures that have broad applications from photonic devices to biomacromolecular recognition. Previous study of evaporative self-assembly has mainly focused on single nonvolatile component systems, and the driving mechanisms have been extensively investigated. In contrast, pattern formation from evaporating multicomponent systems, despite its wide existence in nature and numerous engineering applications, has been rarely explored. In this work, we examine a DNA-colloid binary suspension as a model system to understand the evaporation-induced interfacial hydrodynamics and self-assembled morphology in multicomponent systems involving complex competing intermolecular and interfacial interactions. Direct microscopic observations show that the composition of the binary system plays a critical role in the multiple-ring formation upon evaporation: (1) suspensions with high DNA concentrations and low colloidal concentrations favor the formation of the multiple-ring pattern; (2) the size of colloidal particles added into DNA aqueous droplets can significantly disrupt smooth multiple rings to form rippled rings and curtain-like periodic patterns with a curious spoke-like structure as the size of colloidal particles increases; and (3) the enhancement of DNA-colloid interaction by oppositely charged colloidal particles results in considerably high irregularity of DNA stain ring spacing. We examine the disruption of the multiring morphology under varied conditions and attribute it to local hydrodynamics governed by colloid aggregation and sedimentation. Our results demonstrate the feasibility of fabricating periodic self-assembled hybrid structures via one-step evaporation of droplets consisting of multiple components.