A force-matching method for quantitative hardness measurements by atomic force microscopy with diamond-tipped sapphire cantilevers

We present a new method to improve the accuracy of force application and hardness measurements in hard surfaces by using low-force (<50 μN) nanoindentation technique with a cube-corner diamond tip mounted on an atomic force microscopy (AFM) sapphire cantilever. A force calibration procedure based on the force-matching method, which explicitly includes the tip geometry and the tip-substrate deformation during calibration, is proposed. A computer algorithm to automate this calibration procedure is also made available. The proposed methodology is verified experimentally by conducting AFM nanoindentations on fused quartz, Si(1 0 0) and a 100-nm-thick film of gold deposited on Si(1 0 0). Comparison of experimental results with finite element simulations and literature data yields excellent agreement. In particular, hardness measurements using AFM nanoindentation in fused quartz show a systematic error less than 2% when applying the force-matching method, as opposed to 37% with the standard protocol. Furthermore, the residual impressions left in the different substrates are examined in detail using non-contact AFM imaging with the same diamond probe. The uncertainty of method to measure the projected area of contact at maximum force due to elastic recovery effects is also discussed.     

Characterization of polymer thin films by phase-sensitive acoustic microscopy and atomic force microscopy: a comparative review

The potential of phase-sensitive acoustic microscopy (PSAM) for characterizing polymer thin films is reviewed in comparison to atomic force microscopy (AFM). This comparison is based on results from three-dimensional vector contrast imaging and multimodal imaging using PSAM and AFM, respectively. The similarities and differences between the information that can be derived from the AFM topography and phase images, and the PSAM phase and amplitude micrographs are examined. In particular, the significance of the PSAM phase information for qualitative and quantitative characterization of the polymer films is examined for systems that generate surface waves, and those that do not. The relative merits, limitations and outlook of both techniques, individually, and as a complementary pair, are discussed.     

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.     

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.     

原子力显微镜在生物分子间相互作用力的研究

近年来原子力显微镜在测量生物分子间的相互作用力方面取得显著的进步。本文综述原子力显微镜原理以及在生物分子间相互作用方面的研究,为人们理解分子的识别进程,提供一个新的研究方法。     

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。     

原子力显微镜在聚合物研究中的应用

原子力显微镜以其分辨率高、样品无需特殊制备、实验可在大气环境中进行等优点而广泛应用于聚合物研究之中,弥补扫描隧道显微镜不能观测非导电样品的缺憾。近年来,其应用已由对聚合物表面几何形貌的观测发展到纳米级结构和表面性能的研究领域。在介绍原子力显微镜工作原理的基础上,简要回顾其在聚合物研究方面的若干新应用,并对其应用前景作展望。     

Comparative study of lithium fluoride and graphite by atomic force microscopy (AFM)

The atomic force microscope (AFM) offers the possibility to image the topography of insulating as well as conductive surfaces. Highly oriented pyrolytic graphite (HOPG) was chosen as an example for a layered material and compared to single crystalline lithium fluoride (LiF). Both materials are easily prepared and inert at ambient pressure. Furthermore they are well characterized by Helium atom scattering experiments and other techniques. On HOPG atomic resolution has been achieved. Distortions can be observed which we interpret as a frictional effect. In addition we performed large area scans where we seldomly observed dislocations. For the first time we present measurements on LiF, showing steps of one unit cell height. On larger areas the surface of LiF showed terraces, separated by steps of variable heights, ranging from a few ångströms to 100 Å. We used a static method to get information about the distance dependence of the force between lever and sample. By slowly expanding and retracting the sample piezo and simultaneous measurement of the lever deflection, plots were recorded, showing the force as a function of sample position. The results were compared with theoretical calculations. We could determine the tip radius and found differences between LiF and HOPG being characteristic for the samples.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

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.     

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.     

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.     

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.     

Higher order structure of short immunostimulatory oligonucleotides studied by atomic force microscopy

Immunostimulatory CpG-DNA activates the innate immune system by binding to Toll-like receptor 9. Structurally different CpG-containing oligonucleotides trigger a different type of immune response while activating the same receptor. We therefore investigated the higher order structure of two different classes of immunostimulatory CpG-DNA. Class A, which contains a partly self-complementary sequence and poly-G ends, forms duplexes and nanoparticles in salt solution, while class B, which does not contain these features and is purely linear, does not form a duplex or nanoparticles. Results obtained here by high-resolution atomic force microscopy of classes A and B CpG-DNA, reflect these differences in secondary structure. Detailed structural analysis of the atomic force microscopy topographs is presented for two different sample preparation methods.     

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.     

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。     

Micromorphology analysis of TiO2 thin films by atomic force microscopy images: The influence of postannealing

This work describes an analysis of titanium dioxide (TiO₂) thin films prepared on silicon substrates by direct current (DC) planar magnetron sputtering system in O₂/Ar atmosphere in correlation with three‐dimensional (3D) surface characterization using atomic force microscopy (AFM). The samples were grown at temperatures 200, 300, and 400°C on silicon substrate using the same deposition time (30 min) and were distributed into four groups: Group I (as‐deposited samples), Group II (samples annealed at 200°C), Group III (samples annealed at 300°C), and Group IV (samples annealed at 400°C). AFM images with a size of 0.95 μm × 0.95 μm were recorded with a scanning resolution of 256 × 256 pixels. Stereometric analysis was carried out on the basis of AFM data, and the surface topography was described according to ISO 25178‐2:2012 and American Society of Mechanical Engineers (ASME) B46.1‐2009 standards. The maximum and minimum root mean square roughnesses were observed in surfaces of Group II (Sq = 7.96 ± 0.1 nm) and Group IV (Sq = 3.87 ± 0.1 nm), respectively.     

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.     

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.     

Use of atomic force microscopy (AFM) for microfabric study of cohesive soils

Microfabric reflects the imprints of the geologic and stress history of the soil deposit, the depositional environment and weathering history. Many investigators have been concerned with the fundamental problem of how the engineering properties of clay depend on the microfabric, which can be defined as geometric arrangement of particles within the soil mass. It is believed that scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the only techniques that can reveal particle arrangements of clayey soils directly; however, current research introduces a novel and more advanced technique, atomic force microscopy, to evaluate the microfabric of cohesive materials. The atomic force microscopy has several advantages over SEM/TEM for characterizing cohesive particles at the sub‐micrometre range by providing 3D images and 2D images with Z‐information used in quantitative measurements of soil microfabric using SPIP software, and having the capability of obtaining images in all environments (ambient air, liquids and vacuums). This paper focuses on the use of atomic force microscopy technique to quantify the microfabric of clayey soils by developing the criteria for average and maximum values of angle of particle orientation within the soil mass using proposed empirical equations for intermediate and extreme microfabrics (dispersed, flocculated).