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.     

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.     

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.     

Controlled growth and formation of SAMs investigated by atomic force microscopy

Recently we reported a simple method for obtaining both monolayer thickness and surface patterning using self-assembled monolayers (SAMs). Here we presented a straightforward method for controlling the formation of SAMs over surfaces useful for both chemical and biological applications. Atomic force microscopy (AFM) has been used to investigate the growth mechanism and formation of octadecylsiloxane (ODS) films obtained using a less-reactive silane; octadecyltrimethoxysilane (OTMS). SAMs formation from both OTMS and octadecyltrichlorosilane (ODTS) differ in the hydrolysis step where ODTS results in hydrochloric acid formation, which may affect the delicate features on surfaces. On the other hand, OTMS does not show this behavior. In contrast to monolayer formation from chlorosilane precursors, methoxysilane SAMs have been studied less extensively. Our observations highlight the importance of controlling water content during the formation of ODS monolayers in order to get well-ordered SAMs. We have also seen that, like ODTS, OTMS exhibits monolayer growth through an island expansion process but with a comparatively slow growth rate and different island morphology. The average height of islands, surface coverage, contact angle and root-mean-square (RMS) roughness increase with OTMS adsorption time in a consecutive manner.     

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.     

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.     

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。     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10⁻⁵.     

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.     

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.     

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.     

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al . (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science , 1996, pp. 924–930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.     

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。     

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.     

Dynamic friction measurements with an atomic force microscope on polymer surfaces

The combination of scanning friction force microscopy (SFFM) and lock‐in techniques leads to dynamic SFFM (DSFFM) and provides great advantages in friction force studies with sub‐micrometre resolution. In this paper are presented measurements on thin adsorbed organic films on polymers (polymer blend of 75% poly(allylaminehydrochloride) (PAA) and 25% poly(diallyl‐dimethylammonium chloride) (PDDAC)) and on mica (as a reference). The amplitude and phase response as a function of the excitation amplitude can be explained on hard surfaces by a simple static and dynamic friction model. This model allows us further to distinguish static friction forces and kinetic friction forces in a quantitative way. Furthermore, we demonstrate the use of these spectra to determine the correct modulation amplitude of the excitation to achieve the optimal friction contrasts directly. Polymer data suggest that the viscoelastic shear flow under the atomic force microscope (AFM) tip is responsible for the shape of the phase and amplitude spectrum. Lastly, we demonstrate that DSFFM is a useful technique for surface characterisation in situations where SFFM may not be adequate.     

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.     

Micromechanical and structural properties of a pennate diatom investigated by atomic force microscopy

The mechanisms behind natural nanofabrication of highly structured silicas are increasingly being investigated. We have explored the use of a standard Nanoscope III Multimode atomic force microscope (AFM) to study the silica shell of diatoms. The delicate structures of the shell surface of the diatom Navicula pelliculosa (Bréb.) Hilse were imaged and the shell's micromechanical properties were measured semi-quantitatively with a resolution down to approximately 10 nm. The technique to measure elasticity and hardness with the AFM was demonstrated to be useable even on these hard glass-like surfaces. Different experimental configurations and evaluation methods were tested. They gave a consistent result of the shell micromechanical properties. The first results showed that the diatom shell's overall hardness and elasticity was similar to that of known silicas. However, regions with different mechanical properties were distinguished. The elastic modulus varied from 7 to 20 GPa, from 20 to 100 GPa and from 30 to hundreds of GPa depending on the location. In general, the hardness measurements showed similar spatial differences. The hardness values ranged from 1 to 12 GPa but one specific part of the shell was even harder. Hence, certain localized regions of the shell were significantly harder or more elastic. These regions coincide with known characteristic features and mechanisms appearing at the different stages of the shell's growth. These results show that this method serves as a complementary tool in the study of silica biomineralization, and can detect eventual crystalline phases.     

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.     

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.     

Imaging the native structure of the chaperone protein GroEL without fixation using atomic force microscopy

Most sample preparation methods for scanning probe or electron microscopy require that biomolecules, such as proteins, be fixed. Fixation destroys the molecular functionality and can possibly affect the true molecular structure. Here we report sample preparation conditions that allow the imaging of an unfixed protein, GroEL, under in-vivo conditions, by atomic force microscopy. Under these conditions, the protein should maintain its native structure and biological activity. The typical toroidal shape with pore of the GroEL complex was easily visible in the images. Images of a single complex show dimensions that agree well with crystallographic data. Under in-vivo conditions, it should be possible to study the biological activity and function of proteins.