Effect of aging on the morphology of bitumen by atomic force microscopy

Effect of aging on the morphology of bitumen was investigated. Two bitumens were aged according to the thin film oven test (TFOT), pressure aging vessel (PAV) test and ultraviolet (UV) radiation, respectively. The morphology of the binders before and after aging was characterized by atomic force microscopy. The physical properties and chemical compositions of the binders were also measured. The results showed that aging affected the bitumen morphology significantly. Aging increased the overall surface stiffness of the bitumen and made the bitumen surface more solid-like. The extent of these changes was dependent on aging conditions. TFOT decreased the contrast between the dispersed domains and the matrix, which contributed to the single-phase trend of the binders. The effect of PAV aging on morphology of the binders was dependent on the base bitumen. In one case, it further accelerated the single-phase trend of bitumen in comparison with that after TFOT. In the other case, it caused the phase separation of bitumen. In both cases, PAV aging increased the surface roughness of the binders obviously. As a result of UV aging, the contrast between the matrix phase and dispersed phase was increased due to the difference in sensitivity to UV radiation of the bitumen molecules, which caused or further promoted the phase separation in the binders. Regardless of the aging procedure carried out, a strong correlation was observed between the changes in morphology and physical properties as well as chemical compositions of the binders before and after aging.     

New direct observations of asphalts and asphalt binders by scanning electron microscopy and atomic force microscopy

Observations made using AFM and SEM have been combined in order to study the structure of asphalts. Fluorescence microscopy was used to aid in understanding the structural changes occurring when polymer is added to the asphalts.   With the atomic force microscope we are able to study the structure of the asphalts without any pre-preparation. Despite very low resolution, our study reveal ed a network of asphaltene molecules with regard to asphalt gel. The same result is obtained by SEM observation but with a much better resolution. SEM observation, however, needs an adequate preparation method.   In the presence of polymer we observed a rearrangement of the initial asphaltene association which leads to the assumption that polymer can aggregate the asphaltene phase.     

Bituminous emulsions and their characterization by atomic force microscopy

We present a new method for observing oil-in-water emulsions with a continuous water phase and a dispersed bitumen phase. The fine polydispersed bitumen micelles were adsorbed to an atomically smooth mica substrate and imaged in solution by atomic force microscopy in a liquid cell. The height of the adsorbed bitumen sheet in wet and dry states can be measured and the homogeneity of film formation by coalescence can be determined. Localization of surfactant onto and between bitumen micelles is also visualized.     

C-banding visualized by atomic force microscopy

C-banding is a method used for studying chromosome rearrangements near centromeres and for investigating polymorphisms. In human chromosomes, the C-bands are located at the centromere of all the chromosomes and the distal long arm of the Y chromosome. In this study, we aimed to detect the structural changes in chromosomes during the stages of C-banding by atomic force microscopy. We observed crater-like structures in the chromosomes after 2xSSC (saline sodium citrate) treatment and measured the relative difference between the heights of chromatid and centromere of the chromosomes. Results showed that the relative difference was 3 nm in chromosomes 1, 9, 16, and Y, whereas in the other chromosomes this value was 11.6 nm. After Giemsa staining, the relative difference increased by a factor of 16 in chromosomes 1, 9, 16, and Y. The other chromosomes showed no such increase, which is in accordance with our suggestion that nonhiston proteins associated with DNA in constitutive heterochromatin can make the constitutive heterochromatin resistant to C-banding.     

Low-temperature bitumen stiffness and viscous paraffinic nano- and micro-domains by cryogenic AFM and PDM

In an effort to better understand the structure and behaviour of bitumen in low temperature, we describe the first use of cryogenic atomic force microscopy and phase detection microscopy to characterize bitumen nano‐ and micro‐structures. The results were interpreted in light of glass transition temperatures (T_gs) for bitumen fractions. The domains visible by microscopy, the catana, peri and para phases, were attributed to domains rich in asphaltenes, naphthene and polar aromatics, and saturates, respectively. Between ?10°C and ?30°C, atomic force microscopy images revealed topographic features not visible in atomic force microscopy images acquired at room temperature. According to phase detection microscopy and T_gs, the features were assigned to viscous unfrozen saturates. Upon cooling to ?72°C, unfrozen domains of 20–400 nm were observed. These domains were found in the paraphase rich in saturates and in the periphase rich in naphthene aromatics and polar aromatics. The findings indicate that new viscous domains form upon cooling to low temperatures owing to phase segregation, and that some bitumens are never entirely rigid in low temperatures.     

Adhesion artefacts in atomic force microscopy imaging

Artefacts that affect contrast and arise from adhesion forces in atomic force microscopy images of aramid fibres (both fresh and plasma-treated) are investigated. It is demonstrated that these stem not only from variations in the chemical composition of the surface but also from certain topographical features (which may appear hidden or enhanced in the images), resulting in changes in the lateral forces that are detected by the cantilever and are comparable to the vertical forces. It is also shown that both types of contribution to the forces can be uncoupled to yield images free from these artefacts, thus allowing more accurate quantitative measurements. These artefactual effects are also generally applicable to many other materials.     

Effect of aging on morphology of organo-montmorillonite modified bitumen by atomic force microscopy

The morphology of unmodified and organo-montmorillonite modified bitumens was investigated by atomic force microscopy. The influence of thin film oven test and ultraviolet aging on the morphology of the binders was also analysed. The atomic force microscopy results showed that bitumen displayed a 'bee-like' structure and the dimension of the 'bee-like' structures was decreased to some extent with the introduction of organo-montmorillonite. Organo-montmorillonite showed a better interaction with the dispersed domains in comparison with the matrix in bitumen, which led to an obvious increase in the contrast between the dispersed domains and the matrix in bitumen. Compared with the unmodified bitumen, the single-phase trend in the organo-montmorillonite modified bitumen could be effectively prevented during thin film oven test and ultraviolet aging, indicating its good aging resistance which was in accordance with changes in physical properties of the organo-montmorillonite modified bitumen before and after aging.     

The mechanism of G-banding detected by atomic force microscopy

The morphologic changes occurring in human chromosomes during G-banding by trypsin treatment on the same metaphase were followed with the aid of an atomic force microscope (AFM). It was found that trypsin treatment alone caused a pattern of collapse in the chromosomes that was clearly dependent on the duration of trypsinization. The progressive pattern of collapse first indicated the loss of internal differentiation between chromatids, then bands, and finally all internal structures, except for edges running around the chromosomes' perimeter. When stained with Giemsa, the collapsed chromosomes partly regained their original form, and transverse ridges appeared that correspond to G-positive band regions. However, the treatment of fixed chromosomes with trypsin for 42 s diminished the chromosomal edges, and the z-dimensions could not be measured even with the subsequent application of Giemsa.     

Investigation of nanoparticles by atomic and lateral force microscopy

Metallic nanoparticles have been produced on vitreous carbon substrates by means of thermal evaporation. From pictures of the particles, made by a high-resolution scanning electron microscope (HRSEM), a semispherical shape is suggested due to the total mass of deposited material. Atomic force microscopy (AFM) has been applied to this sample in order to get direct topographic information. The AFM has been operated with normal and super tips, the latter having a smaller cone angle and radius, thus following more precisely the contours of an object. Simultaneously lateral-force microscopic (LFM) images have been recorded. Major differences between the contents of HRSEM- and AFM-images are considered, emphasizing the important influence of the tips' geometry. Both the AFM and LFM line scans have been compared with and have qualitatively agreed with those calculated under simplifying assumptions.     

Frictional effects in atomic force microscopy of Langmuir-Blodgett films

Frictional effects in atomic force microscopy (AFM) of Langmuir-Blodgett films of 1, 2-dipalmitoyl-snglycero-phosphoglycerol were examined. Height measurements of the Langmuir layers are strongly influenced by the orientation of the cantilevers used in AFM relative to the sample. A simple model is used to describe the frictional effects and to calculate the real height of the monolayers.     

Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.     

Imaging DNA molecules on mica surface by atomic force microscopy in air and in liquid

DNA molecules immobilized on mica surface by various methods have been observed by atomic force microscopy both in air and in liquid. Divalent cations and 3-aminopropyltriethoxysilane (APTES) modified mica surface have been used to immobilize the DNA molecules. Optimal DNA and divalent cations concentration for AFM imaging are presented. Among the different methods of modifying mica surface with APTES, the water solution modifying method appears to get the best results. When using high DNA concentration for AFM imaging, DNA networks can be formed. A simple method to extend long DNA molecules is demonstrated. The optimal imaging conditions and AFM operating techniques are discussed. Different DNA immobilizing methods have been compared and evaluated.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

3-D morphological characterization of the liver parenchyma by atomic force microscopy and by scanning electron microscopy

A comparative study of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging of the healthy human liver parenchyma was carried out to determine the similarities and the differences. In this study, we compared the fine hepatic structures as observed by SEM and AFM. Although AFM revealed such typical hepatic structures as bile canaliculi and hepatocytes, it also showed the location of the nucleus and chromatin granules in rough relief structure, which was not visible by SEM. By contrast, SEM visualized other structures, such as microvilli, the central vein, and collagenous fibers, none of which was visualized by AFM. For better orientation and confirmation of most of the structures imaged by SEM and AFM, Congo Red-stained specimens were also examined. Amyloid deposits in the Disse's spaces were shown especially clearly in these images. The differences between the SEM and AFM images reflected the characteristics of the detection systems and methods used for sample preparation. Our results reveal that more detailed information on hepatic morphology is obtained by exploiting the advantages of both SEM and AFM.     

Scanning tunnelling and atomic force microscopy performed with the same probe in one unit

The technique demonstrated here provides features of both scanning tunnelling microscopy (STM) and atomic force microscopy (AFM). The metallic probe acts to record current variations and sense forces from the same sample area simultaneously. Thus, separate images may be recorded, in registry. The collected data allows real space correlations between some electrical properties and the geometric structure of a sample surface. The same tip is used since the geometry and condition of the tip can effect the data recordings. Platinum alloys, tungsten and graphite tips have been employed successfully. An AFM lever can respond to surface contact forces, within the elastic limits of the sample, while electric current is sensed by the tip of the lever. The usefulness of this experimental procedure is tested here by an application to semiconducting samples of Ag-doped CdTe in air and in paraffin oil media.     

Membrane deformation of living glial cells using atomic force microscopy

Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epi-fluorescence illumination to monitor the presence of fluo-3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force–distance curves indicated that standard tips did not penetrate the membrane while sharper Supertips^TM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.     

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.     

Imaging and nano-dissection of tobacco mosaic virus by atomic force microscopy

Tobacco mosaic virus (TMV) has been deposited on freshly cleaved mica substrates. The topography was investigated by contact, non-contact and lateral-force microscopy under ambient conditions in air. The results were in accord with known dimensions of TMV (i.e. 18 nm in diameter and 300 nm in length). However, convolution of tip shape with TMV morphology resulted in an apparent width of 80–140 nm in the lateral plane, a factor of 4–7 greater than the known diameter. Other artefacts - broadening and double images - were observed and ascribed to tip anomalies. High force loadings and slow repetitive scanning resulted in controlled removal of parts of the TMV structure. Accordingly, it was possible to reveal and image the central core channel of the TMV. The precision and resolution of dissection induced by AFM is currently limited by the shape of the tip, having a 40-nm radius of curvature for standard Si₃N₄ tips. It is estimated that sharper tips, with a radius of curvature of less than 10 nm, should be able to resolve, non-destructively, the protein subunits in the non-contact mode, and selectively remove single subunits in the contact mode.     

Combined atomic force and scanning reflection interference contrast microscopy

A sphere attached to a cantilever is used simultaneously as an atomic force microscope (AFM) tip and as a curved reflective surface for producing scanning reflection interference contrast microscope (RICM) images of fluorescent beads dried onto a glass slide. The AFM and RICM images are acquired in direct registration which enables the identification of individually excited beads in the AFM images. The addition of a sharp, electron beam-deposited tip to the sphere gives nanometer resolution AFM images without loss of optical contrast.     

Fast contact-mode atomic force microscopy on biological specimen by model-based control

The dynamic behavior of the piezoelectric tube scanner limits the imaging rate in atomic force microscopy (AFM). In order to compensate for the lateral dynamics of the scanning piezo a model based open-loop controller is implemented into a commercial AFM system. Additionally, our new control strategy employing a model-based two-degrees-of-freedom controller improves the performance in the vertical direction, which is important for high-speed topographical imaging. The combination of both controllers in lateral and vertical direction compensates the three-dimensional dynamics of the AFM system and reduces artifacts that are induced by the systems dynamic behavior at high scan rates. We demonstrate this improvement by comparing the performance of the model-based controlled AFM to the uncompensated and standard PI-controlled system when imaging pUC 18 plasmid DNA in air as well as in a liquid environment.