Surface properties of Aspergillus oryzae spores investigated by atomic force microscopy

Spores of the filamentous fungus Aspergillus oryzae have a great biotechnological potential for the production of highly active proteins. To date, little is known about the molecular mechanisms of spore aggregation, a phenomenon observed during germination in liquid medium. Here, atomic force microscopy (AFM) imaging and force measurements were used to characterize, under aqueous conditions, the surface morphology and macromolecular interactions of A. oryzae spores in relation to their aggregation behavior. Dormant spores were covered with a discontinuous layer of about 35 nm thickness, as revealed by height images. High-resolution deflection images showed that this layer consisted of rodlets, 10±1 nm in diameter, that were assembled in parallel to form fascicles interlaced with different orientations. The germinating spore surface was much rougher and showed streaks oriented in the scanning direction, indicating that the probe was interacting with soft material. Retraction force curves were strikingly different depending on the spore physiological state: while dormant spores exhibited non-adhesive properties, germinating spores showed single or multiple attractive forces of 400±100 pN magnitude, along with characteristic elongation forces and rupture lengths ranging from 20 to 500 nm. These elongation forces are attributed to the stretching of long, flexible cell surface macromolecules and suggested to play a role in the aggregation process by promoting bridging interactions.     

Predation, death, and survival in a biofilm: Bdellovibrio investigated by atomic force microscopy

Biofilms are complex microbial communities that are resistant to attack by bacteriophages and to removal by drugs and chemicals. Here we use atomic force microscopy (AFM) to image the attack on Escherichia coli biofilms by Bdellovibrio bacteriovorus 109J. Bdellovibrio is a small, predatory bacterium that invades and devours other Gram-negative bacteria. We demonstrate that under dilute nutrient conditions, bdellovibrios can prevent the formation of simple bacterial biofilms and destroy established biofilms; under richer conditions the prey bacteria persist and are not eradicated, but may be shifted toward solution populations. Using AFM we explore these bacterial interactions with more detail and accuracy than available by more traditional staining assays or optical microscopy. AFM also allows us to investigate the nanoscale morphological changes of the predator, especially those related to motility. This demonstration of Bdellovibrio's successful predation in a biofilm inspires us to consider ways that it might be used productively for industrial, medical, agricultural, and biodefensive purposes.     

Surface structure of thin asymmetric PS-b-PMMA diblock copolymers investigated by atomic force microscopy

Asymmetric poly(styrene-b-methyl methacrylate) (PS-b-PMMA) diblock copolymers of molecular weight M_n = 29,700 g mol⁻¹ (M_PS = 9300 g mol⁻¹M_PMMA = 20,100 g mol⁻¹, PD = 1.15, χ_PS = 0.323, χ_PMMA = 0.677) and M_n = 63,900 g mol⁻¹ (M_PS = 50,500 g mol⁻¹, M_PMMA = 13,400 g mol⁻¹, PD = 1.18, χ_PS = 0.790, χ_PMMA = 0.210) were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization. Atomic force microscopy (AFM) was used to investigate the surface structure of thin films, prepared by spin-coating the diblock copolymers on a silicon substrate. We show that the nanostructure of the diblock copolymer depends on the molecular weight and volume fraction of the diblock copolymers. We observed a perpendicular lamellar structure for the high molar mass sample and a hexagonal-packed cylindrical patterning for the lower molar mass one. Small-angle X-ray scattering investigation of these samples without annealing did not reveal any ordered structure. Annealing of PS-b-PMMA samples at 160 °C for 24 h led to a change in surface structure.     

Investigating the morphology and mechanical properties of blastomeres with atomic force microscopy

Atomic force microscopy (AFM) was used to directly investigate the morphology and mechanical properties of blastomeres during the embryo development. With AFM imaging, the surface topography of blastomeres from two‐cell, four‐cell, and eight‐cell stages was visualized, and the AFM images clearly revealed the blastomere's morphological changes during the different embryo developmental stages. The section measurements of the AFM topography images of the blastomeres showed that the axis of the embryos nearly kept constant during the two‐cell, four‐cell, and eight‐cell stages. With AFM indenting, the mechanical properties of living blastomeres from several embryos were measured quantitatively under physiological conditions. The results of mechanical properties measurements indicated that the Young's modulus of the two blastomeres from two‐cell embryo was different from each other, and the four blastomeres from the four‐cell embryo also had variable Young's modulus. Besides, the blastomeres from two‐cell embryos were significantly harder than blastomeres from four‐cell embryos. These results can improve our understanding of the embryo development from the view of cell mechanics. Copyright © 2013 John Wiley & Sons, Ltd.     

Surface morphology of antifrictional polymer materials: Experience in atomic force and electron microscopy

The morphology of the friction surface of antifrictional charged carbon plastics is investigated by atomic force and scanning electron microscopy. Methodical approaches to composites probe preparing are described, and possibilities of methods for examination of friction surface are shown. On the basis of the obtained results, the role of nanosized modifiers of various chemical natures (used in manufacturing antifrictional materials) is highlighted, and the mechanisms of the processes that occur in carbon plastics on the tribological contact are suggested.     

Interaction of small amounts of bovine serum albumin with phospholipid monolayers investigated by surface pressure and atomic force microscopy

The influence of small amounts of bovine serum albumin (BSA) (nM concentration) on the lateral organization of phospholipid monolayers at the air-water interface and transferred onto solid substrates as one-layer Langmuir-Blodgett (LB) films was investigated. The kinetics of adsorption of BSA onto the phospholipid monolayers was monitored with surface pressure isotherms in a Langmuir trough, for the zwitterionic dipalmitoylphosphatidyl ethanolamine (N,N-dimethyl-PE) and the anionic dimyristoylphosphatidic acid (DMPA). A monolayer of N,N-dimethyl-PE or DMPA incorporating BSA was transferred onto a solid substrate using the Langmuir-Blodgett technique. Atomic force microscopy (AFM) images of one-layer LB films displayed protein-phospholipid domains, whose morphology was characterized using dynamic scaling theories to calculate roughness exponents. For DMPA-BSA films the surface is characteristic of self-affine fractals, which may be described with the Kardar-Parisi-Zhang (KPZ) equation. On the other hand, for N,N-dimethyl-PE-BSA films, the results indicate a relatively flat surface within the globule. The height profile and the number and size of globules varied with the type of phospholipid. The overall results, from kinetics of adsorption on Langmuir monolayers and surface morphology in LB films, could be interpreted in terms of the higher affinity of BSA to the anionic DMPA than to the zwitterionic N,N-dimethyl-PE. Furthermore, the effects from such small amounts of BSA in the monolayer point to a cooperative response of DMPA and N,N-dimethyl-PE monolayers to the protein.     

Spectral force analysis using atomic force microscopy reveals the importance of surface heterogeneity in bacterial and colloid adhesion to engineered surfaces

Coatings developed to reduce biofouling of engineered surfaces do not always perform as expected based on their native properties. One reason is that a relatively small number of highly adhesive sites, or the heterogeneity of the coated surface, may control the overall response of the system to initial bacterial deposition. It is shown here using an approach we call spectral force analysis (SFA), based on force volume imaging of the surface with atomic force microscopy, that the behavior of surfaces and coatings can be better understood relative to bacterial adhesion. The application of vapor deposited TiO₂ metal oxide increased bacterial and colloid adhesion, but coating the surface with silica oxide reduced adhesion in a manner consistent with SFA based on analysis of the “stickiest” sites. Application of a TiO₂-based paint to a surface produced a relatively non-fouling surface. Addition of a hydrophilic layer coating to this surface should have decreased fouling. However, it was observed that this coating actually increased fouling. Using SFA it was shown that the reason for the increased adhesion of bacteria and particles to the hydrophilic layer was that the surface produced by this coating was highly heterogeneous, resulting in a small number of sites that created a stickier surface. These results show that while it is important to manufacture surfaces with coatings that are relatively non-adhesive to bacteria, it is also essential that these coatings have a highly uniform surface chemistry.     

Surface morphology of polypyrrole core/polyacrolein shell latex by atomic force microscopy (AFM)

 Polypyrrole latex (P(P)), synthesized in Redox polymerization of pyrrole, was used as seed for radical polymerization of acrolein initiated with K₂S₂O₈. In this process the polypyrrole core/polyacrolein shell latex (P(P–A)) was obtained. Morphology of the surface of P(P–A) particles was investigated by atomic force microscopy (AFM). It was found that macromolecules of polyacrolein are not randomly distributed on the surface of polypyrrole but form patches. Apparently, attraction between macromolecules of poly-acrolein in the surface layer is high and the enthalpy of formation of polyacrolein macromolecule clusters is sufficient to compensate, at least, the negative entropy change due to ordering of these macromolecules. Thickness of the polyacrolein layer on the surface of polypyrrole particles, which were covered only partially with polyacrolein, was equal to only 1.6 nm (standard deviation σ= 0.2 nm) and thus, it was reasonable to assume that it corresponded to the monolayer coverage. Received: 30 April 1997 Accepted: 25 August 1997     

Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy

Analyte–wall interaction is a significant problem in capillary electrophoresis (CE) as it may compromise separation efficiencies and migration time repeatability. In CE, self-assembled polyelectrolyte multilayer films of Polybrene (PB) and dextran sulfate (DS) or poly(vinylsulfonic acid) (PVS) have been used to coat the capillary inner wall and thereby prevent analyte adsorption. In this study, atomic force microscopy (AFM) was employed to investigate the layer thickness and surface morphology of monolayer (PB), bilayer, (PB-DS and PB-PVS), and trilayer (PB-DS-PB and PB-PVS-PB) coatings on glass surfaces. AFM nanoshaving experiments providing height distributions demonstrated that the coating procedures led to average layer thicknesses between 1 nm (PB) and 5 nm (PB-DS-PB), suggesting the individual polyelectrolytes adhere flat on the silica surface. Investigation of the surface morphology of the different coatings by AFM revealed that the PB coating does not completely cover the silica surface, whereas full coverage was observed for the trilayer coatings. The DS-containing coatings appeared on average 1 nm thicker than the corresponding PVS-containing coatings, which could be attributed to the molecular structure of the anionic polymers applied. Upon exposure to the basic protein cytochrome c, AFM measurements showed an increase of the layer thickness for bare (3.1 nm) and PB-DS-coated (4.6 nm) silica, indicating substantial protein adsorption. In contrast, a very small or no increase of the layer thickness was observed for the PB and PB-DS-PB coatings, demonstrating their effectiveness against protein adsorption. The AFM results are consistent with earlier obtained CE data obtained for proteins using the same polyelectrolyte coatings.     

Observation of banded spherulites and lamellar structures by atomic force microscopy

Spherulites are common structures of semi-crystalline polymers. It has been known that semi-crystalline polymers can form spherulites when crystallized from solution or from melt. A dark Maltese cross of a spherulite could be easily observed under the polarized optical microscopy (POM). Moreover, some spherulites show an additional alternating dark and bright concentric ring structure that is attributed to the regular twisting of the radial crystallite ribbons as they grow from the spherulit…     

Label-free quantification of Tacrolimus in biological samples by atomic force microscopy

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose–response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.     

Observation of adhesive force and energy of adsorbents on rubber substrates by atomic force microscopy

We observed the surface morphology and adhesive interaction of adsorbents on rubber substrates by atomic force microscopy (AFM). The detachment of adsorbents from rubber substrates is an important issue for various machines like home appliances and laundry machine. Since a clean surface of the functioning parts is required, a frequent cleaning process must be developed. In particular, dust and lint have a tendency to bind to the rubber surface of a laundry machine. Several practical methods have been attempted to remove these particles from the surface. Pure water, detergent, sodium hypochlorite (65 °C), and cold water (18 °C) are treated onto artificial dust and lint mixtures on rubber with water fluid by rapid rpm. The dust‐and‐lint adsorbents are investigated by AFM after the treatment, and topographic images and force–distance (F–D) curves were generated for the samples. The roughness, measured as the root mean square, is a key factor to judge the cleaning quality. From the F–D curves, we are able to obtain adhesive energy in addition to adhesive force which will yield qualitative measures of the interactions between the remaining adsorbents and the rubber surface. Considering the values that were measured, hot water with water fluid by rapid rpm offers the best performance when cleaning the surface. The chemical like sodium hypochlorite is good for thinning the materials, but it solidifies them, which is eventually detrimental to proper functioning. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative study of filled rubber microstructure by optical and atomic force microscopy

A comprehensive approach is proposed for studying the microstructure of filled rubbers by optical and atomic force microscopy (AFM). The optical results are found to be dependent on the illumination angle. Algorithms based on the mathematical morphology are developed for the processing of optical images (removing scratches, identifying agglomerates). AFM-images are treated by a segmentation method which separates a continuous surface into segments that match filler. Parameters of secondary filler structures and the size of an area of homogeneous filler dispersion are obtained from the analysis of the segmented images. Seven carbon black filled rubbers with different mixing times are investigated. The combination of AFM with optical imaging techniques makes it possible to perform a quantitative structural analysis at scales from tens of nanometers to tens of microns, and to establish the relationship between the mixing time and the filler microstructure over the whole range of filler peculiarities.     

Nanocharacterization and nanofabrication of a Nafion thin film in liquids by atomic force microscopy

We demonstrated the nanocharacterization and nanofabrication of a Nafion thin film using atomic force microscopy (AFM). AFM images showed that the Nafion molecules form nanoclusters in water, in 5% methanol, and in acetic acid. Young's modulus E of a Nafion film was estimated by sequential force curve measurements in water and in 5% methanol on one sample surface. Ewater/E5% methanol was 1.75 +/- 0.40, so the film was much softer in 5% methanol than in water. Even when solvent was replaced from 5% methanol to water, Young's modulus was not recovered soon. We showed the first example of the mechanical properties of a Nafion film on the nanoscale. Furthermore, we succeeded in fabricating 3D nanostructures on a Nafion surface by AFM nanolithography in liquids. Our results showed the new potential of the AFM nanolithography of a polymer film by softening the molecules in liquids.     

Looking at the micro-topography of polished and blasted Ti-based biomaterials using atomic force microscopy and contact angle goniometry

Surface topography of polished and blasted samples of a Ti6Al4V biomaterial has been studied using an atomic force microscope. Surface RMS roughness and surface area have been measured at different scales, from 1 to 50 μm, while at distances below 10 μm the surface RMS roughness in both kinds of samples is not very different, this difference becomes significant at larger scanning sizes. This means that the surface roughness scale that could have a main role in cell adhesion varies depending on the size, shape and flexibility of participating cells. This consideration suggests that in cell–material interaction studies, surface roughness should not be considered as an absolute and independent property of the material, but should be measured at scales in the order of the cell sizes, at least if a microscopic interpretation of the influence of roughness on the adhesion is intended. The microscopic information is contrasted with that coming from a macroscopic approach obtained by contact angle measurements for polar and non-polar liquids whose surface tension is comprised in a broad range. Despite the very large differences of contact angles among liquids for each surface condition, a similar increase for the blasted surface with respect to the polished has been found. Interpretation of these results are in accordance with the microscopic analysis done through the use of a functional roughness parameter, namely the valley fluid retention index, evaluated from the AFM images, which has been shown not to correlate with the RMS roughness, one of the most commonly used roughness parameter.     

Looking at the micro-topography of polished and blasted Ti-based biomaterials using atomic force microscopy and contact angle goniometry

Surface topography of polished and blasted samples of a Ti6Al4V biomaterial has been studied using an atomic force microscope. Surface RMS roughness and surface area have been measured at different scales, from 1 to 50 μm, while at distances below 10 μm the surface RMS roughness in both kinds of samples is not very different, this difference becomes significant at larger scanning sizes. This means that the surface roughness scale that could have a main role in cell adhesion varies depending on the size, shape and flexibility of participating cells. This consideration suggests that in cell–material interaction studies, surface roughness should not be considered as an absolute and independent property of the material, but should be measured at scales in the order of the cell sizes, at least if a microscopic interpretation of the influence of roughness on the adhesion is intended. The microscopic information is contrasted with that coming from a macroscopic approach obtained by contact angle measurements for polar and non-polar liquids whose surface tension is comprised in a broad range. Despite the very large differences of contact angles among liquids for each surface condition, a similar increase for the blasted surface with respect to the polished has been found. Interpretation of these results are in accordance with the microscopic analysis done through the use of a functional roughness parameter, namely the valley fluid retention index, evaluated from the AFM images, which has been shown not to correlate with the RMS roughness, one of the most commonly used roughness parameter.     

Dependency of particle sizes and colloidal stability of polyelectrolyte complex dispersions on polyanion structure and preparation mode investigated by dynamic light scattering and atomic force microscopy

Polyelectrolyte complex (PEC) dispersions were prepared by controlled mixing of three random copolymers of sodium 2-acrylamido-2-methylpropanesulfonate (AMPS) with either t-butyl acrylamide (TBA) [P(AMPS54-co-TBA46) and P(AMPS37-co-TBA63)] or methyl methacrylate (MM) [P(AMPS52-co-MM48)] with an ionene-type polycation, containing 95 mol % N,N-dimethyl-2-hydroxypropyleneammonium chloride repeat units (PCA5), with their structural characteristics being deeply investigated by dynamic light scattering (DLS) and atomic force microscopy (AFM). Shape, size, and polydispersity of the PEC dispersions were directly observed by AFM as a function of polyanion structure, the ratio between charges, n-/n+, and the titrant addition rate (TAR). The particle sizes increased and the colloidal stability decreased with the increase of the nonionic comonomer content and with the decrease of TAR. It was demonstrated that the medium particle sizes of the complex nanoparticles adsorbed on silicon wafers measured by AFM, in the dry state, were close but always lower than those measured by DLS, both before and after the complex stoichiometry.     

Features of the hedritic morphology of β‐isotactic polypropylene studied by atomic force microscopy

The lamellar organization of melt‐crystallized β‐isotactic polypropylene was studied by atomic force microscopy (AFM) after permanganic etching. Hedritic objects grown at a high crystallization temperature (140–143 °C) were investigated. Essential features of the hedritic development were revealed by the characteristic projections exposed at the sample surface. A three‐dimensional view of the morphology was obtained by AFM. Hedritic growth proceeded mainly by branching around screw dislocations resulting in new lamellae that further developed. Successive lamellar layers often diverged. Deviation from the planar lamellar habit was observed, varying with the position within the hedrite. Twisting of the lamellae also was observed occasionally in the vicinity of the screw dislocations. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 672–681, 2000     

Comparative characterisation by atomic force microscopy and ellipsometry of soft and solid thin films

Ellipsometry and atomic force microscopy (AFM) were used to study the film thickness and the surface roughness of both ‘soft’ and solid thin films. ‘Soft’ polymer thin films of polystyrene and poly(styrene–ethylene/butylene–styrene) block copolymer were prepared by spin‐coating onto planar silicon wafers. Ellipsometric parameters were fitted by the Cauchy approach using a two‐layer model with planar boundaries between the layers. The smooth surfaces of the prepared polymer films were confirmed by AFM. There is good agreement between AFM and ellipsometry in the 80–130 nm thickness range. Semiconductor surfaces (Si) obtained by anisotropic chemical etching were investigated as an example of a randomly rough surface. To define roughness parameters by ellipsometry, the top rough layers were treated as thin films according to the Bruggeman effective medium approximation (BEMA). Surface roughness values measured by AFM and ellipsometry show the same tendency of increasing roughness with increased etching time, although AFM results depend on the used window size. The combined use of both methods appears to offer the most comprehensive route to quantitative surface roughness characterisation of solid films. Copyright © 2007 John Wiley & Sons, Ltd.