High-resolution Topographic Images Research Articles

Abstract: S3-5 HUMAN LCAT DEFICIENCY

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.

Tip-enhanced near-field optical microscopy of carbon nanotubes

We review recent experimental studies on single-walled carbon nanotubes on substrates using tip-enhanced near-field optical microscopy (TENOM). High-resolution optical and topographic imaging with sub 15 nm spatial resolution is shown to provide novel insights into the spectroscopic properties of these nanoscale materials. In the case of semiconducting nanotubes, the simultaneous observation of Raman scattering and photoluminescence (PL) is possible, enabling a direct correlation between vibrational and electronic properties on the nanoscale. So far, applications of TENOM have focused on the spectroscopy of localized phonon modes, local band energy renormalizations induced by charge carrier doping, the environmental sensitivity of nanotube PL, and inter-nanotube energy transfer. At the end of this review we discuss the remaining limitations and challenges in this field.

Imaging and Detection of Single Molecule Recognition Events on Organic Semiconductor Surfaces

The combination of organic thin film transistors and biological molecules could open new approaches for the detection and measurement of properties of biological entities. To generate specific addressable binding sites on such substrates, it is necessary to determine how single biological molecules, capable of serving as such binding sites behave upon attachment to semiconductor surfaces. Here, we use a combination of high-resolution atomic force microscopy topographical imaging and single molecule force spectroscopy (TREC), to study the functionality of antibiotin antibodies upon adsorption on pentacene islands, using biotin-functionalized, magnetically coated AFM tips. The antibodies could be stably adsorbed on the pentacene, preserving their functionality of recognizing biotin over the whole observation time of more than one hour. We have resolved individual antigen binding sites on single antibodies for the first time. This highlights the resolution capacity of the technique.

Mechanical properties of porous methyl silsesquioxane and nanoclustering silica films using atomic force microscope

Mechanical properties of porous methyl silsesquioxane samples with dielectric constant 2.4 and 2.0 and a recently developed nanoclustering silica film samples with dielectric constants 2.3 and 2.0 were evaluated using an atomic force microscope based nanoindentation. It was found that the Young’s modulus and the hardness decrease while the fracture toughness increases with a decrease in the dielectric constant in the same type of material. Moreover, the Young’s modulus and the hardness of the nanoclustering silica films were observed to be at least twice and fracture toughness values ~1.3–1.5 higher than those for methyl silsesquioxane films with similar dielectric constants. The high resolution topographic imaging capability of atomic force microscope was shown to be particularly useful in the measurement of cracks generated by the ultra-low indentation loads, and the evaluation of the fracture toughness of the nanoscale volumes of materials.

A stabler non contact atomic force microscopy imaging using a tuning fork for air and liquid environments: The zero phase mode atomic force microscopy

We describe a method to perform noncontact atomic force microscopy (NC-AFM) in liquid and in air using a quartz crystal tuning fork. The latter, thanks to its increased stability originating from its higher stiffness, compared to cantilevers, enables very low oscillation amplitudes and thus, should yield a very high sensitivity to the force interactions. However, applying conventional NC-AFM technique fails, due to the phase lock loop demodulator becoming unstable as the input signal to noise ratio decreases when lowering the oscillation amplitude. Therefore we have developed so-called zero phase mode atomic force microscopy based on phase modulation. Comparisons with conventional frequency modulation mode using the same mechanical setup demonstrate gains in sensitivity and in scanning rates. High resolution topographic images, as well as simultaneous dissipation images can be recorded using this very convenient and effective technique.

Local work function measurements of epitaxial graphene

The work function difference between single layer and bilayer graphene grown epitaxially on 6H-SiC(0001) has been determined to be 135±9 meV by means of the Kelvin probe force microscopy. Bilayer films are found to increase the work function as compared to single layer films. This method allows an unambiguous distinction between interface layer, single layer, and bilayer graphene. In combination with high-resolution topographic imaging, the complex step structure of epitaxial graphene on SiC can be resolved with respect to substrate and graphene layer steps.

Temperature-dependent imaging of living cells by AFM

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure–function relationships in living cells. Lipid–lipid and lipid–protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 °C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.

Application of Terrestrial Laser Scanning in determining the pattern of late Pleistocene and Holocene fault displacement from the offset of pluvial lake shorelines in the Alvord extensional basin, northern Great Basin, USA

The northwardtrending Alvord extensional basin of southeastern Oregon lies along the northern margin of the Great Basin. The basin is nearly 200 km long and 15 km wide and is bound and internally dissected by a complex system of active normal faults. The faults cut Pleistocene wave-cut terraces that formed along successive shorelines of pluvial Lake Alvord. The wave-cut terraces are incised into late Tertiary volcanic and sedimentary rocks and unconsolidated Quaternary sediments. The terraces formed during stillstands of the ancient lake, during at least two cycles of lake fill and desiccation in the Pleistocene. Wave-cut terraces are divided into two sets, with the topographically higher and older Serrano terrace series consisting of three shorelines and the Alvord terrace series composed of the five topographically lower and younger shorelines. Shorelines are dated locally, and together with regional correlation to other pluvial lakes, the Serrano highstand is estimated as 200–130 ka and the Alvord highstand as 20–15 ka. High-resolution topographic images of the terrace morphology acquired by Terrestrial Laser Scanning georeferenced with the Global Positioning System allowed detailed analysis of shoreline altitudes where they are crosscut by faults. Variation in shoreline altitude measured across faults and on opposing sides of the basin indicates that fault slip occurred during and following periods of lake-level recession. The Serrano terrace highstand records a cumulative vertical displacement of 137.5 ± 3.6 m and the vertical offset of the younger Alvord terrace series highstand is 72.5 ± 2.8 m. Fault displacement is heterogeneously distributed across the basin. Faults along the western margin of the basin accommodated nearly 50% of the total displacement, with one fault accommodating over 20% of the total displacement budget. The residual displacement is distributed across the basin. As much as 30% of the cumulative offset is taken up by structures concealed beneath basin cover and 20% by structures exposed in the highlands along the eastern flank of the basin. Since formation of the Alvord shorelines, the rate of fault displacement was nonperiodic and the basin underwent elevated activity in the late Pleistocene and early Holocene. Vertical displacement rates vary through time, and offset of Alvord terraces occurred at 3.6–7.3 mm/yr, whereas displacement of the Serrano terraces ranged from 0.7 to 1.1 mm/yr. When the horizontal component of motion is calculated by using fault dips of 60°, the 104 yr time-scale rate determined from the Alvord terraces is as much as 2.4–4.2 mm/yr and exceeds the contemporary horizontal displacement across the basin of 1.75 mm/yr determined geodetically. The 105 yr horizontal displacement rate calculated for the Serrano highstand is substantially lower, 0.4–0.6 mm/yr. Spatial and temporal pattern of faulting within the Alvord basin illustrates the complexity of strain release within the basin and highlights the length- and time-scale dependence of deformation rate estimation within extensional basins.

Topography of metallic surfaces subjected to plastic strain: Roughness, spatial correlations, and eigenvalue spectral entropy

This paper describes an analysis technique that integrates high-resolution topographical imaging and rigorous matrix-based three-dimensional characterization methods. We employ scanning confocal laser microscopy to obtain topographic images of the surface of an aluminum alloy subjected to various levels of uniaxial plastic strain. These images are discretized into a square array of pixels, each of which is then assigned a numerical value corresponding to the deviation of surface height relative to an averaged background. The result is a set of real, non-Hermitian n×n matrices, which are diagonalized to produce a collection of spectra, each of which consists of n complex eigenvalues. These eigenvalue spectra are observed to change systematically as the degree of plastic strain is varied. Because this approach is based solely on the behavior of the eigenvalue spectra, it eliminates the need for the a priori assumptions about surface character used in conventional topographic analyses. The information contained within an eigenvalue spectrum is distilled into a scalar measure of topographic disorder, referred to as the “spectral entropy.” The spectral entropy is observed to decrease monotonically with increasing plastic strain. This behavior is consistent with the observed topographical changes induced by plastic strain. In addition, the spectral entropy can be decomposed into a constant term that is independent of all spatial correlations that occur in the surface roughness and a term that incorporates these correlations at all levels of complexity.

Study of the ZDDP Antiwear Tribofilm Formed on the DLC Coating Using AFM and XPS Techniques

Abstract To meet the challenge of the increasing demand of fuel economy, in recent years low friction nonferrous coatings such as diamond-like carbon (DLC) coatings have become very popular for automotive tribo-components. The interaction of lubricant additives, which are designed for ferrous surfaces, with nonferrous coatings is an important issue for the automotive and lubricant industries. The aim of this paper is to establish a link between the evolution of antiwear zinc dialkyl dithiophosphate (ZDDP) tribofilm and the tribological performance of a DLC coating under boundary lubrication conditions. Experiments were performed in a pin-on-plate reciprocating tribotester to produce the tribofilm. Atomic force microscopy (AFM) was used to record high resolution topographical images of the ZDDP films while chemical analysis of the ZDDP tribofilms was performed using X-ray photoelectron spectroscopy (XPS). Results in this study show that the ZDDP tribofilm consists of short chain zinc pyrophosphate (Zn2P2O7) and zinc metaphosphate (ZnOP2O5), and it is formed along the raised portion of the initial marks of the DLC surface.

Nanoscale chemistry and mechanical properties of tribofilms on Al–Si alloy (A383): interaction of ZDDP, calcium detergent and molybdenum friction modifier

The interaction of a friction modifier and a calcium phenate detergent additive, with zinc dialkyl dithiophosphates (ZDDPs) in the formation of antiwear films on A383, has been studied using synchrotron radiation and nanoindentation techniques. X-ray absorption near edge structure (XANES) spectroscopy has shown that films prepared from oils containing both ZDDP and detergent, and ZDDP and molybdenum dithiocarbamate (MoDTC), are chemically similar to, but thicker than those made from oils containing only ZDDP. In addition, wear was greatly reduced in the presence of the detergent which was correlated with the basicity and the presence of the friction modifier. The phosphorus K and L edge XANES spectra show that the tribofilms are polyphosphate glasses of similar nature to those found on steel, but characterised by a shorter chain length. The sulphur K edge shows a MoS2 like film and under certain conditions, the presence of a sulphate species is detected. High resolution topographic images and mechanical properties were determined by atomic force microscopy and imaging nanoindentation. The films formed in the presence of the detergent exhibited similar mechanical responses independent of the conditions tested. The indentation modulus of the films on the Al matrix always appear much softer than the films formed on the Si grains whether or not the lubricant contains only ZDDP, or both ZDDP and MoDTC.

‘‘Extraordinary’’ Surface Phase Transition at (100) Surface of NbSe3

We have investigated both q1 and q2 charge-density wave transitions occuring in NbSe3 by means of low temperature scanning tunneling microscopy under ultra-high vacuum on in-situ cleaved (100) surface. High-resolution topographical images with molecular resolution were obtained in the temperature range between 5K and 140K. We studied the temperature dependency of the order parameters of both transitions using Fourier transform of the STM images. Our results show that at the free (100) NbSe3 surface, the low temperature q2 CDW transition occurs above 70K, i.e. more than 10K above the bulk transition temperature reported by x-ray diffraction experiment or by tunneling junction measurements involving an unfree NbSe3 surface. We propose that as q2 phonon modes in NbSe3possess a component transverse to the surface, they might be softer in the top layer than in the bulk, leading to an extraordinary phase transition for the q2 CDW at surface. Additionnally, at variance with previously reported STM measurements, our images show a bias voltage polarity dependency that could be interpreted in a lower density of states on Se atoms of chain II for the occupied states than for the empty states.

Single Molecule Force Microscopy on Cells and Biological Membranes

Atomic force microscopy (AFM) enables high resolution topographic imaging of biological samples under near-physiological conditions. Therefore, the AFM is optimally suited for investigation of biological membranes and cell surfaces, as exemplified by studies on bacterial S-layers, purple membranes and cultured living cells. Topographic imaging allows visualizing single proteins and protein assemblies in native membranes, as well as substructures of live cells, such as cytoskeletal architecture. In addition to high-resolution imaging, the measurement of mechanical forces yields detailed insight into structure-function relationships of molecular processes in their native environment. In molecular recognition force microscopy, interaction forces between tip-bound ligands and membrane-embedded receptors can be studied under well-controlled buffer conditions and effectors concentrations. In case of low lateral density and inhomogeneous distribution of the target molecules in a cell membrane, fluorescence microscopy can help to guide the AFM tip to the membrane proteins of interest, which can subsequently be investigated by molecular recognition force microscopy. Keywords: Atomic force microscopy (AFM), Force spectroscopy, Molecular recognition, Recognition imaging, Molecular forces, Cell membranes, Membrane proteins

Imaging Mechanical Properties of Living Cells by Scanning Probe Microscopy

Scanning probe microscope (SPM) has been developed as a powerful tool for obtaining high resolution topographic images of biological samples in their natural aqueous environment. SPM can also be used to evaluate mechanical properties because its probe is physically in contact with the samples during measurement. To obtain cellular stiffness with SPM, we have proposed two methods: a force modulation mode and a force mapping mode. Considering the influence of the drag force of liquids, we have successfully improved the quantitative evaluation of cellular stiffness by using the force modulation mode. Experiments performed using the two methods revealed that the local stiffness of fibroblasts was not homogeneous on the cell surface but largely varied from point to point. It was revealed that spatial and temporal distributions of cellular stiffness originate in cytoskeletal distribution, mode of cellular migration, and intracellular contractile force.

Structural and Compositional Mapping of a Phase-Separated Langmuir−Blodgett Monolayer by Atomic Force Microscopy

The structure and composition of a phase-separated arachidic acid (C19H39COOH) (AA) and perfluorotetradecanoic acid (C13F27COOH) (PA) Langmuir-Blodgett monolayer film was characterized by several different types of atomic force microscopic measurements. At the liquid-air interface, surface pressure-area isotherms show that mixtures of the two acids follow the additivity rule expected from ideal mixtures. Topographic images of the deposited monolayer indicate that the surfactants are oriented normal to the substrate surface, and that the acids undergo phase separation to form a series of discontinuous, hexagonal domains separated by a continuous domain. A combination of lateral force (friction) imaging and adhesion force measurements show that the discontinuous domains are enriched in AA, whereas the surrounding continuous domain is a mixture of both AA and PA. This was further verified by selective, in situ dissolution of AA by n-hexadecane, followed by high-resolution topographical imaging of the discontinuous domains.

Scanning tunneling spectroscopy of single-strand deoxyribonucleic acid for sequencing

The authors performed scanning tunneling spectroscopy (STS) analysis of single-strand deoxyribonucleic acid (DNA) with fluorescein isothiocyanate (FITC) dye at the 5′ terminal (FITC-5′-agctgtac-3′). They obtained high-resolution topographic scanning tunneling microscope images of the DNA that showed eight nucleotide bases and one FITC molecule. The current versus voltage (I-V) data of FITC had local maxima or plateaus, whereas those of DNA had simple growth curves. They also found that in the STS analysis, the I-V data of the DNA were fitted well by an exponential curve in the positive and negative bias regions. They also discuss the possibility of whether four nucleotides (adenine, guanine, cytosine, and thymine) can be differentiated through STS experiments.

Long polymeric tips of atomic force microscopy for large biological samples

We show a new atomic force microscopy technique for obtaining high-resolution topographic images of large bio-samples. To obtain high-resolution topographic images for the samples, we fabricated a long polymeric tip with a small protrusion using two-photon adsorbed photo-polymerization techniques. The obtained tip length was over 50 microm, and the tip was used directly to visualize COS-1 and 293 cells. Compared with commercial tips, the long tip made it easier to obtain topographic images of the large cells. In the magnified topographic images, the sub-100-nm resolution was confirmed with the long tips. This long probe tip is expected to broaden large sample-related studies and applications in the future.

Characterisation of pitting corrosion by white light interferometry

An improved method is described for obtaining detailed information on the size and shape of many corrosion pits by examination with White Light Interferometry. The entire surface is first imaged at low resolution by automatically stitching individual images into one composite image. Specially written software analyses the image and determines the location of all corrosion pits. The program then guides the white light interferometer in making high resolution topographic images of each pit. The images are analyzed to give any desired data about the pit geometry, such as depth, maximum width, projected area, volume, etc. Curves showing the depth distribution for each pit can be generated along with detailed topographic images of all pits. The technique does not work for pits that undercut the surface, and there is a practical size limit of about 20 cm 2. The method is therefore best suited for studying the early stages of pit propagation.

A variable temperature mechanical analysis of ZDDP-derived antiwear films formed on 52100 steel

The nanomechanical properties of antiwear films formed from zinc dialkyl-dithiophosphates (ZDDPs) on steel have been studied by nanoindentation techniques as a function of temperature. X-ray absorption P K- and L- near edge structure (XANES) spectroscopy has shown that films prepared from oils containing ZDDPs on 52100 steel (pin on flat coupons) consist primarily of medium chain polyphosphates with sulphur (S K-edge) predominantly present as sulphide. Using various scanning probe techniques, high-resolution topographic images and mechanical properties can be extracted at the same length scale. Using focused ion beam (FIB) milling we have compared real cross-sectional film thickness with a value estimated from the P K-edge XANES. We report the first measurements of the elastic modulus of the antiwear films at elevated temperatures relevant to the automobile operating conditions ( T ≤ 200 °C). The antiwear films demonstrated a relatively constant indentation modulus over a wide range of temperatures consistent with their efficacy in reducing wear by preventing asperity contact.

High-Resolution X-ray Topography of Dislocations in 4H-SiC Epilayers

AbstractSilicon carbide (SiC) substrates and epilayers contain many crystal defects, such as micropipes, screw dislocations, threading edge dislocations (TEDs), basal plane dislocations (BPDs) and stacking faults. To investigate these defects, synchrotron radiation topography is frequently carried out. When the monochromatic synchrotron X-ray topography is taken by the grazing-incidence reflection geometry using 11-28 reflection, screw dislocations, TEDs and BPDs are simultaneously seen and shown as different topographic images [1]. Many studies of dislocations were reported using 11-28 reflections in 4H-SiC [1,2]. Topographic images of the dislocations have been analyzed by the ray-tracing method of computer simulation [3]. However, experimental images of dislocations were not fully matched to the fine structure of simulation images, because of a lack of resolution in recording media: conventional films and nuclear emulsion plates [3]. This time, we report obtaining high-resolution topographic images using a new recording medium, and compare results between the experiment and the computer simulation. Synchrotron topography in 11-28 reflection was carried out at SPring8 applying holography films as high-resolution recording media. The TED images are distinguished as four types, which have ribbon-like features with different rotating angles, through the use of the films. The four different TED images agree well with the computer simulated images which have been reported by Vetter et.al. taking into account of the different Burgers vector directions [3]. By comparing the three topographic images taken at g=-12-18, 11-28 and 2-1-18, we confirmed experimentally that the four types of TED images originated from the difference of Burgers vector directions. We also investigated high-resolution topographic images of elementary screw dislocations, micropipes, and BPDs in 4H-SiC epilayers. The experimental image of screw dislocation fairly matched with simulated image. The fine features in the experimental topographic images of micropipes and BPDs are also compared with the simulated images in detail. [1] T. Ohno, H. Yamaguchi, S. Kuroda, K. Kojima, T. Suzuki, K. Arai: J. cryst. Growth. Vol. 260 (2004) 209. [2] H. Tsuchida, T. Miyanagi, I. Kamata, T. Nakamura, R. Ishii, K. Nakayama and Y.Sugawara: Jpn. J. Appl. Phys. Vol. 25, (2005), L806-808. [3] W. Vetter, H. Tsuchida, I. Kamata, M. Dudley: J. Appl. Cryst. Vol. 38, (2005), 442-447.