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.     

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.     

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.     

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.     

Promises and problems of biological atomic force microscopy

Recent developments in the biological applications of atomic force microscopy are reviewed, and the great potential of this novel, high-resolution imaging technique, including its limitations and possible future directions for biological research, are discussed.     

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。     

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs.     

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.     

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip–sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase–distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever–sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.     

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.     

Study of SU-8 photoresist cross-linking process by atomic force acoustic microscopy

In this paper, a method is presented to detect the different phases of epoxy cross-linking process and the subsurface structures of SU-8 thin films by atomic force acoustic microscopy (AFAM). The AFAM imaging of SU-8 thin films was investigated under different exposure and bake conditions. Optimized conditions were obtained for the cross-linking of SU-8 thin film at the exposure does of eight laser pulses with the laser fluence 10 mJ cm^–2 per pulse and the post exposure bake (PEB) time at 90 s. The subsurface structures of undeveloped SU-8 thin films were visible in the AFAM images. This method provides an effective and low-cost way for the determination of different phases of epoxy cross-linking process in nanostructured compounds, for the non-destructive testing of subsurface defects, and for the evaluation of the quality of patterned structures.     

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.     

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.     

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。     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

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.     

原子力显微镜在生物医学中的应用

原子力显微镜 (AFM)是近十几年来表面成像技术中最重要的进展之一。它具有非常高的分辨率。本文将阐述原子力显微镜的工作原理 ,分析原子力显微镜在生物医学中的应用现状 ,包括生物医学样品的表面形貌观测 ,在液体中的观测 ,生物分子之间力谱曲线的观测 ,以及生物医学样品制备技术等。     

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.     

Real-time atomic force microscopy in lubrication condition

We have studied frictional force and wear problem in real-time atomic force microscopy in contact-mode using a resonator type mechanical scanner allegedly reported. The fast scanning may cause wear in the sample surface or the tip, and may deteriorate the image quality. Mineral oil was used to make a lubricious surface on a polycarbonate sample, and it was found that the interfacial frictional force was decreased. A Si tip which was coated with a hydrophobic film by means of chemical modification was confirmed to diminish the frictional force in the fast scanning process. The resultant image quality was improved due to reduced friction and wear.     

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.