Velocity dependent friction laws in contact mode atomic force microscopy

Friction forces in the tip–sample contact govern the dynamics of contact mode atomic force microscopy. In ambient conditions typical contact radii between tip and sample are in the order of a few nanometers. In order to account for the large interaction area the dynamics of contact mode atomic force microscope (AFM) is investigated under the assumption of a multi-asperity contact interface between tip and sample. Thus, the kinetic friction force between tip and sample is the product of the real contact area between both solids and the interfacial shear strength. The velocity strengthening of the lateral force is modeled assuming a logarithmic relationship between shear-strength and velocity. Numerical simulations of the system dynamics with this empirical model show the existence of two different regimes in contact mode AFM: steady sliding and stick–slip where the tip undergoes periodically stiction and kinetic friction. The state of the system depends on the scan velocity as well as on the velocity dependence of the interfacial friction force between tip and sample. Already small viscous damping contributions in the tip–sample contact are sufficient to suppress stick–slip oscillations.     

原子力显微镜原理与应用技术

本文简述原子力显微镜的工作原理,对比说明敲击模式的优越性,指出针尖-样品卷积效应和假象产生的原因,并例证其应用领域及其测试效果。     

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.     

Novel operation mode for eliminating influence of inclination angle and friction in atomic force microscopy

The accuracy of topography imaging in contact force mode of atomic force microscopy (AFM) depends on the one-to-one corresponding relationship between the cantilever deflection and the tip–sample distance, whereas such a relationship cannot be always achieved in the presence of friction and incline angle of sample surface. Recently, we have developed a novel operation mode in which we keep the van der Waals force as constant instead of the applied normal force, to eliminate the effect of inclination angle and friction on topography imaging in the contact force mode. We have improved our AFM to enable the new operation mode for validation. Comparative experiments have been performed and the results have shown that the effect of friction and inclination angle on topography imaging in contact mode of AFM can be eliminated or at least decreased effectively by working in the new operation mode we present.     

A symmetrical stretching stage for electrical atomic force microscopy

We present a remotely-controlled device for sample stretching, designed for use with atomic force microscopy (AFM) and providing electrical connection to the sample. Such a device enables nanoscale investigation of electrical properties of thin gold films deposited on polydimethylsiloxane (PDMS) substrate as a function of the elongation of the structure. Stretching and releasing is remotely controlled with use of a dc actuator. Moreover, the sample is stretched symmetrically, which gives an opportunity to perform AFM scans in the same site without a time-consuming finding procedure. Electrical connections to the sample are also provided, enabling Kelvin probe force microscopy (KPFM) investigations. Additionally, we present results of AFM imaging using the stretching stage.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

Contrasting static-to-kinetic friction transitions on layers of an autophobically dewetted polymer film using Fourier-analyzed shear modulation force microscopy

Fourier analysis of oscillating forces at a laterally modulated tip provides new insight into static-to-kinetic friction transitions on ultrathin polyvinyl alcohol (PVA) films. In addition to contrast in sliding friction, layers of autophobically dewetted PVA films exhibit remarkable contrast in the transition from static to kinetic friction as derived from spatially resolved Fourier analysis. These differences relate to strong adsorption of first layer to mica substrate and concomitant conformational arrest, as compared to bulk-like behavior in the second layer. The third Fourier harmonic is found to be a sensitive gauge to variable degrees of sliding as a function of both lateral drive amplitude (0.25–25 nm) and normal load (tensile to compressive). For a 2.5-nm drive on PVA, it is discovered that a largely static contact at compressive loads becomes a largely sliding contact at tensile loads. This finding has implications for the analysis of shear modulation force microscopy of polymers in the context of contact mechanics models, and for studies under variable sample compliance as a function of temperature or plasticizer absorption.     

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1–4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1–2°. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.     

原子力显微镜在多糖分子结构研究中的应用

评述原子力显微镜在多糖分子结构和功能研究的进展,AFM不仅可以在空气和液体中对多糖分子单分子和聚集体成像,得到单分子的直径、长度等量化信息和分子聚集体形貌特征。近年来AFM还用于在液体池中操纵单个多糖分子,获取单分子力学谱研究分子的弹性与构型转变的关系,在单分子水平上对多糖进行鉴定,用于细胞表面大分子黏附作用和细胞识别的研究等。AFM新技术的不断出现,必将在高分子科学的研究中起到越来越重要的作用。     

Ribosome substructure investigated by scanning force microscopy and image processing

Scanning force microscopy was used to study the ultrastructure of eukaryotic ribosomes from Chironomus pallidivittatus in the polysomal complex. Positively stained polysomes were imaged, and the resulting three-dimensional ribosomal structures were further processed by statistical analyses of virtual cross-sections parallel to the substrate plane. Structural investigations were based on parameters which are minimally influenced by the tip geometry, like section plane centre or axis ratio. In the lower part of the structure a shift of the section centres was observed, suggesting an attached structure. The geometry of the sections revealed an elliptical shape in the upper part (axis ratio 1.52 ± 0.22), with a less elongated shape in the lower region (axis ratio 1.41 ± 0.18). A model for the surface topography of a positively stained ribosome exhibiting a small subunit attached along the long side of an elliptical large structure is proposed.     

Climbing the steps of viral atomic force microscopy: visualization of Dengue virus particles

In recent years, the application of atomic force microscopy (AFM) to biological systems has highlighted the potential of this technology. AFM provides insights into studies of biological structures and interactions and can also identify and characterize a large panel of pathogens, including viruses. The Flaviviridae family contains a number of viruses that are important human and animal pathogens. Among them, Dengue virus causes epidemics with fatal outcomes mainly in the tropics. In this study, Dengue virus is visualized for the first time using the in air AFM technique. Images were obtained from a potassium-tartrate gradient-purified virus. This study enhances the application of AFM as a novel tool for the visualization and characterization of virus particles. Because flavivirus members are closely related, studies of the morphologic structure of the Dengue virus can reveal strategies that may be useful to identify and study other important viruses in the family, including the West Nile virus.     

日本精工SAP400原子力显微镜性能的改进

日本精工公司(SEIKO)生产的原子力显微镜的功能多、性能好,国内已经有近百台的拥有量。它的信号放大、处理主要由锁相放大器完成。但其内部的锁相放大器灵敏度不够高,外加一台高性能、高灵敏度的锁相放大器,可大大提高其性能。用美国斯坦福公司产的SR830锁相放大器对本实验室的日本SEIKO公司SPA400原子力显微镜进行性能改进,可使其性能有很大提高。     

Spectroscopy and atomic force microscopy of biomass

Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.     

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.     

Ultrasonic force microscopy: detection and imaging of ultra-thin molecular domains

The analysis of the formation of ultra-thin organic films is a very important issue. In fact, it is known that the properties of organic light emitting diodes and field effect transistors are strongly affected by the early growth stages. For instance, in the case of sexithiophene, the presence of domains made of molecules with the backbone parallel to the substrate surface has been indirectly evidenced by photoluminescence spectroscopy and confocal microscopy. On the contrary, conventional scanning force microscopy both in contact and intermittent contact modes have failed to detect such domains. In this paper, we show that Ultrasonic Force Microscopy (UFM), sensitive to nanomechanical properties, allows one to directly identify the structure of sub-monolayer thick films. Sexithiophene flat domains have been imaged for the first time with nanometer scale spatial resolution. A comparison with lateral force and intermittent contact modes has been carried out in order to explain the origins of the UFM contrast and its advantages. In particular, it indicates that UFM is highly suitable for investigations where high sensitivity to material properties, low specimen damage and high spatial resolution are required.     

Quantitative analysis of surface micro-roughness alterations in human spermatozoa using atomic force microscopy

A new male contraceptive given the name RISUG^® (an acronym for Reversible Inhibition of Sperm Under Guidance) has been developed by our research group. RISUG^® is a bioactive polymer and is injected into the lumen of the vas deferens using a no-scalpel approach. The polyelectrolytic nature of this contraceptive induces a surface charge imbalance on sperm membrane system leading to its destabilization. Complete disintegration of the plasma membrane with subsequent rupture and dispersion of the acrosomal contents is observed on RISUG^® treatment. In the present study, micro-structural properties of human spermatozoa exposed to RISUG^® in vitro have been quantitatively analysed using atomic force microscopy. The parameters used to quantify these morphological changes include amplitude (peak–valley height difference, arithmetic roughness, root mean square roughness) and spatial roughness. Factor loadings (Varimax rotation) have been used to determine the parameters displaying maximum variation. Further, sperm cells have been classified in various principal-component planes using principal-component analysis. The periodic structural features of the atomic force microscopy images have also been obtained using power spectral analysis.     

Multifrequency high-speed phase-modulation atomic force microscopy in liquids

We have developed a new technique, called multifrequency high-speed phase-modulation atomic force microscopy (PM-AFM) in constant-amplitude (CA) mode based on the simultaneous excitation of the first two flexural modes of a cantilever. By performing a theoretical investigation, we have found that this technique enables the simultaneous imaging of the surface topography, energy dissipation and elasticity (nonlinear mapping) of materials. We experimentally demonstrated high-speed imaging at a scan speed of 5 frames/s for a polystyrene (PS) and polyisobutylene (PIB) polymer-blend thin-film surface in water.     

QD as a bifunctional cell-surface marker for both fluorescence and atomic force microscopy

Fluorescent quantum dots (QDs) are a new class of fluorescent label and have been extensively used in cell imaging. Streptavidin-conjugated QDs have a diameter of ca. 10–15 nm; therefore when used as probes to label cell-surface biomolecules, they can provide contrast enhancement under atomic force microscopy (AFM) and allow specific proteins to be distinguished from the background. In addition, the size and fluorescent properties potentially make them as probes in correlative fluorescence microscopy (FM) and AFM. In this study, we tested the feasibility of using QD-streptavidin conjugates as probes to label wheat germ agglutinin (WGA) receptors on the membrane of human red blood cells (RBCs) and simultaneously obtain fluorescence and AFM images. The results show that the distribution of QDs labeled on human RBCs was non-uniform and that the number of labeled QDs on different erythrocytes varied significantly, which perhaps indicates different ages of the erythrocytes. Thus, QDs may be employed as bifunctional cell-surface markers for both FM and AFM to quantitatively investigate the distribution and expression of membrane proteins or receptors on cell surface.     

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.     

Capturing the elasticity and morphology of live fibroblast cell cultures during degradation with atomic force microscopy

Atomic force microscopy, in a liquid environment, was used to capture in vitro the morphological and mechanical changes that cultured fibroblasts undergo as time elapses from the completion of the cell culture. Topography images illustrated that initially, the nucleus had a height of 1.18 ± 0.2 μm, and after 48 h it had decreased to 550 ± 60 nm; similarly, the cell membrane exhibited significant shrinkage from 34 ± 4 to 23 ± 2 μm. After each image scan, atomic force microscopy indentation was performed on the centre of the nucleus, to measure the changes in the cell elasticity. Examination of the force‐distance curves indicated that the membrane elastic modulus at the nucleus remained the same within the time frame of 48 h, even though the cell morphology had significantly changed.