Determination of Coniothyrium minitans conidial and germling lectin avidity by flow cytometry and digital microscopy

The avidity of conidia and 48-h-old germlings of Coniothyrium minitans for FITC-conjugated lectins was characterised by flow cytometry and digital microscopy. Six isolates of C. minitans representing three morphological types were compared. Binding of Con A, SBA and WGA by conidial populations varied markedly in extent and pattern between isolates, however, with increasing culture age, conidia from all isolates demonstrated a significant reduction in lectin avidity. Germling isolates bound significantly different amounts of lectins and lectin binding differed significantly with locality. Spore walls of all germlings from all isolates bound more ConA compared with hyphal apices and mature hyphal walls. In contrast, hyphal apices of the majority of germling isolates, readily bound SBA and mature hyphal walls of germling isolates bound more WGA than other regions of the germlings. Such differential lectin binding by conidia and germlings may influence their specific surface interactions and adherence characteristics.

Microscopy imaging of liposomes:from coverslips to environmental SEM

A plethora of techniques for the imaging of liposomes and other bilayer vesicles are available. However, sample preparation and the technique chosen should be carefully considered in conjunction with the information required. For example, larger vesicles such as multilamellar and giant unilamellar vesicles can be viewed using light microscopy and whilst vesicle confirmation and size prior to additional physical characterisations or more detailed microscopy can be undertaken, the technique is limited in terms of resolution. To consider the options available for visualising liposome-based systems, a wide range of microscopy techniques are described and discussed here: these include light, fluorescence and confocal microscopy and various electron microscopy techniques such as transmission, cryo, freeze fracture and environmental scanning electron microscopy. Their application, advantages and disadvantages are reviewed with regard to their use in analysis of lipid vesicles.

Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes. Copyright © Informa Healthcare.

Analysis of powder caking in multicomponent powders using atomic force microscopy to examine particle properties

Atomic force microscopy has been used to study the surface properties of model spray dried powders. Phase imaging, nanoindentation and force modulation microscopy have differentiated between the different surface material properties of the particles, revealing a regular dispersion of soft, oil rich areas distributed across the particles' surface. Humidity and temperature cycling effects on the caking behavior of the particles have also been investigated, with significant morphology changes and onset of caking found to occur within relatively short periods of time.

Caking behaviour of spray-dried milk powders:using scanning probe microscopy to study nanoscale surface properties and material composition

Spray drying is widely used to manufacture many powdered products, with the drying process parameters having significant influence over the final powder's surface properties and propensity for unwanted caking. In most cases caking experiments are performed on bulk powders, but especially in multi-component powders, it is often difficult to interpret these results, where interaction effects between particles can be complex. Here the technique of scanning probe microscopy is used to characterize the nanoscale properties of spray dried model milk powders in order to investigate the surface properties of the powders.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

The use of atomic force microscopy in investigating particle caking mechanisms

Spray-dried materials are being used increasingly in industries such as food, detergent and pharmaceutical manufacture. Spray-dried sodium carbonate is an important product that has a great propensity to cake; its moisture-sorption properties are very different to the crystalline and amorphous species, with a great affinity for atmospheric moisture. This work demonstrates how the noncontact surface analysis of individual particles using atomic force microscopy can highlight the possible mechanisms of unwanted agglomeration. The nondestructive nature of this method allows cycling of localised humidity in situ and repeated scanning of the same particle area. The resulting topography and phase scans showed that humidity cycling caused changes in the distribution of material phases that were not solely dependent on topographical changes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On chlorosulphonation and the electron microscopy of polyethylene

It is shown that chlorosulphonation is a major aid to the electron microscopy of polyethylene for various samples which had mostly been crystallized at high pressures and included at least a proportion of the so-called chain-extended form. It is confirmed that sheets of excess electron density are produced at lamellar surfaces, but also including lateral surfaces. This is due primarily to the incorporation of chlorine and sulphur rather than to added uranium. The time to achieve an overall reaction varies sensitively with morphology, decreasing as the number of diffusion channels increases. Crystallinity is gradually lost, but sufficient crystals remain when a sample has become uniform, and in their initial orientations, for diffraction studies to be possible. The technique has been used to demonstrate that, during melt crystallization, the thickness of one lamella changes in response to altered growth conditions. This is direct confirmation that lamellar thickness is determined by secondary nucleation at the growth front. The tapered profile of a growing lamella previously observed in thick crystals of various polymers has been observed for chain-folded polyethylene lamellae, providing further evidence that this is a general feature of melt growth. © 1977 Chapman and Hall Ltd.

In situ aberration corrected-transmission electron microscopy of magnesium oxide nanocatalysts for biodiesels

Biofuels are promising renewable energy sources and can be derived from vegetable oil feedstocks. Although solid catalysts show great promise in plant oil triglyceride transesterification to biodiesel, the identification of active sites and operating surface nanostructures created during their processing is essential for the development of efficient heterogeneous catalysts. Systematic, direct observations of dynamic MgO nanocatalysts from a magnesium hydroxide-methoxide precursor were performed under controlled calcination conditions using novel in situ aberration corrected-transmission electron microscopy at the 0.1 nm level and quantified with catalytic reactivity and physico-chemical studies. Surface structural modifications and the evolution of extended atomic scale glide defects implicate coplanar anion vacancies in active sites in the transesterification of triglycerides to biodiesel. The linear correlation between surface defect density (and therefore polarisability) and activity affords a simple means to fine tune new, energy efficient nanocatalysts for biofuel synthesis. © 2009 Springer Science+Business Media, LLC.

Deformation microstructure under nanoindentations in Cu using 3D x-ray structural microscopy

We have used a recently developed x-ray structural microscopy technique to make nondestructive, submicron-resolution measurements of the deformation microstructure below a 100mN maximum load Berkovich nanoindent in single crystal Cu. Direct observations of plastic deformation under the indent were obtained using a ~0.5 µm polychromatic microbeam and diffracted beam depth profiling to make micron-resolution spatially-resolved x-ray Laue diffraction measurements. The local lattice rotations underneath the nanoindent were found to be heterogeneous in nature as revealed by geometrically necessary dislocation (GND) densities determined for positions along lines beneath a flat indent face and under the sharp Berkovich indent blade edges. Measurements of the local rotation-axes and misorientation-angles along these lines are discussed in terms of crystallographic slip systems.

Characterization of Acanthamoeba-microsphere association by multiparameter flow cytometry and confocal microscopy

Background: Acanthamoebae, in common with other protozoa, readily endocytose particulate material, which in turn may lead to the spread of infectious disease. Methods: Evaluation and quantification of plain and carboxylate FITC-microsphere association with acanthamoebal trophzoites was undertaken using a combination of flow cytometry and confocal microscopy. Trophozoites from strains and species of Acanthamoeba were exposed to plain and carboxylate FITC-microspheres. Microsphere size and aspects such as trophozoite starvation, maturity, and exposure to metabolic inhibitors were assessed. Results: All species and strains of Acanthamoeba readily endocytosed plain and carboxylate microspheres. Starving trophozoites significantly increased binding and potential ingestion of microspheres, whereas trophozoites of increasing maturity lost such abilities. Trophozoites showed a significant preference for 2.0- and 3.0-μm-diameter microspheres when compared with other sizes, which in turn could occupy much of the cytoplasm. The physiological inhibitors sodium azide, 2,4-clinitrophenol, and cytochalasin B reduced microsphere association with trophozoites; however, some microspheres still bound and associated with trophozoites after inhibitor exposure, a manifestation of both active and inactive agent involvement in microsphere endocytosis. Conclusions: Even though the origins of microsphere binding by acanthamoebal trophozoite remains shrouded, the combination of flow cytometry and confocal microscopy supported synergistic quantification and qualification of trophozoite-microsphere endocytosis. © 2006 International Society for Analytical Cytology.

Directed phase separation of PFO:PS blends during spin-coating using feedback controlled in situ stroboscopic fluorescence microscopy

Uniform thin-films of polymer blends can be produced through spin-coating, which is used on an industrial scale for the production of light emitting diodes, and more recently organic photovoltaic devices. Here, we present the results of the direct observation, and control, over the phase separation of polystyrene and poly(9,9′-dioctylfluorene) during spin-coating using high speed stroboscopic fluorescence microscopy. This new approach, imaging the fluorescence, from a blend of fluorescent + non-fluorescent polymers allows for intensity to be directly mapped to composition, providing a direct determination of composition fluctuations during the spin-coating process. We have studied the compositional development and corresponding structural development for a range of compositions, which produce a range of different phase separated morphologies. We initially observe domains formed by spinodal decomposition, coarsening via Ostwald Ripening until an interfacial instability causes break-up of the bicontinuous morphology. Ostwald ripening continues, and depending upon composition a bicontinuous morphology is re-established. By observing compositional and morphological development in real-time, we are able to direct and control morphological structure development through control of the spin coating parameters via in situ feedback. © 2013 The Royal Society of Chemistry.

Multicolour nonlinearly bound chirped dissipative solitons

The dissipative soliton regime is one of the most advanced ways to generate high-energy femtosecond pulses in mode-locked lasers. On the other hand, the stimulated Raman scattering in a fibre laser may convert the excess energy out of the coherent dissipative soliton to a noisy Raman pulse, thus limiting its energy. Here we demonstrate that intracavity feedback provided by re-injection of a Raman pulse into the laser cavity leads to formation of a coherent Raman dissipative soliton. Together, a dissipative soliton and a Raman dissipative soliton (of the first and second orders) form a two (three)-colour stable complex with higher total energy and broader spectrum than those of the dissipative soliton alone. Numerous applications can benefit from this approach, including frequency comb spectroscopy, transmission lines, seeding femtosecond parametric amplifiers, enhancement cavities and multiphoton fluorescence microscopy.