Optimizing AC-mode atomic force microscope imaging

We have operated an atomic force microscope in ambient air with several oscillating cantilever modes to establish the optimal scanning parameters to maximize image resolution and to minimize probe and sample damage. This was done by scanning a surface in air and correlating scan parameters such as oscillation amplitude and damping with image resolution. We also examined the geometry of the probe with a scanning electron microscope, before and after scanning, in order to determine whether the scanning technique had an effect on the geometry of the probe tip. If the probe is oscillated such that it contacts the surface on each oscillation, substantial damage or “wear” to the probe occurs and significant degradation of image quality was observed. In ambient air, the optimal conditions are achieved when the probe penetrates the contamination layer and reverses direction before touching the surface. Under these “near-contact” conditions no probe damage is observed and high-image resolution can be maintained indefinitely.     

原子力显微镜的力曲线分析与转化

原子力显微镜测定的力曲线需转化为力位移曲线来应用.力位移曲线是以任意点为零点的,当研究粘附或者分子模型对比时,需要知道针尖样品间的作用力或确切的零点位置,这时需将其转化为力-距离曲线.本文首先从力曲线的测定原理得出了典型的力曲线,之后从理论上分析了力曲线、力位移曲线和力-距离曲线间的转化,从中得出了转化过程中需要的两个重要参量:灵敏度和零距离,并提出了确定方法.最后,利用MATLAB实现了曲线的自动转化.     

Calibration of the scanning (atomic) force microscope with gold particles

Scanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.     

Quantification of the lateral detachment force for bacterial cells using atomic force microscope and centrifugation

To determine the lateral detachment force for individual bacterial cells, a quantitative method using the contact mode of an atomic force microscope (AFM) was developed in this study. Three key factors for the proposed method, i.e. scan size, scan rate and cantilever choice, were evaluated and optimized. The scan size of 40×40 μm² was optimal for capturing sufficient number of adhered cells in a microscopic field and provide adequate information for cell identification and detachment force measurement. The scan rate affected the measurement results significantly, and was optimized at 40 μm/s considering both force measurement accuracy and experimental efficiency. The hardness of applied cantilevers also influenced force determination. The proposed protocol for cantilever selection is to use those with the lowest spring constant first and then step up to a harder cantilever until all cells are detached. The lateral detachment force of Escherichia coli cells on polished stainless steel and a glass-slide coated with poly-l-lysine were measured as 0.763±0.167 and 0.639±0.136 nN, respectively. The results showed that the established method had good repeatability and sensitivity to various bacteria/substrata combinations. The detachment force quantified by AFM (0.639±0.136 nN) was comparable to that measured by the centrifugation method (1.12 nN).     

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.     

Sampling effects influence heights measured with atomic force microscopy

The atomic force microscope (AFM) is an exquisitely delicate probe measuring the height of a specimen at discrete sampling points in a fixed two‐dimensional (2D) raster. The resulting topograph is a 2D digital image, with each pixel representing a distinct height measurement. The height of an object is determined as the average of the maximum heights measured above the supporting surface. We show that such object heights derived from a variety of organic samples depend critically on the sampling or pixel size of the 2D raster. It is concluded that to obtain accurate specimen heights, the pixel size must be small enough to resolve submolecular structures and thus ensure representative sampling of the height variation on the surface.     

Application of atomic force microscopy to the study of natural and model soil particles

The structure and surface chemistry of soil particles has extensive impact on many bulk scale properties and processes of soil systems and consequently the environments that they support. There are a number of physiochemical mechanisms that operate at the nanoscale which affect the soil's capability to maintain native vegetation and crops; this includes soil hydrophobicity and the soil's capacity to hold water and nutrients. The present study used atomic force microscopy in a novel approach to provide unique insight into the nanoscale properties of natural soil particles that control the physiochemical interaction of material within the soil column. There have been few atomic force microscopy studies of soil, perhaps a reflection of the heterogeneous nature of the system. The present study adopted an imaging and force measurement research strategy that accounted for the heterogeneity and used model systems to aid interpretation. The surface roughness of natural soil particles increased with depth in the soil column a consequence of the attachment of organic material within the crevices of the soil particles. The roughness root mean square calculated from ten 25 microm(2) images for five different soil particles from a Netherlands soil was 53.0 nm, 68.0 nm, 92.2 nm and 106.4 nm for the respective soil depths of 0-10 cm, 10-20 cm, 20-30 cm and 30-40 cm. A novel analysis method of atomic force microscopy phase images based on phase angle distribution across a surface was used to interpret the nanoscale distribution of organic material attached to natural and model soil particles. Phase angle distributions obtained from phase images of model surfaces were found to be bimodal, indicating multiple layers of material, which changed with the concentration of adsorbed humic acid. Phase angle distributions obtained from phase images of natural soil particles indicated a trend of decreasing surface coverage with increasing depth in the soil column. This was consistent with previous macroscopic determination of the proportions of organic material chemically extracted from bulk samples of the soils from which specimen particles were drawn. Interaction forces were measured between atomic force microscopy cantilever tips (Si(3)N(4)) and natural soil and model surfaces. Adhesion forces at humic acid free specimen surfaces (Av. 20.0 nN), which are primarily hydrophilic and whose interactions are subject to a significant contribution from the capillary forces, were found to be larger than those of specimen surfaces with adsorbed humic acid (Av. 6.5 nN). This suggests that adsorbed humic acid increased surface hydrophobicity. The magnitude and distribution of adhesion forces between atomic force microscopy tips and the natural particle surfaces was affected by both local surface roughness and the presence of adsorbed organic material. The present study has correlated nanoscale measurements with established macroscale methods of soil study. Thus, the research demonstrates that atomic force microscopy is an important addition to soil science that permits a multiscale analysis of the multifactorial phenomena of soil hydrophobicity and wetting.     

光学原子力显微镜中的蒙特卡罗方法

扫描探针显微镜(Scanning probe microscopy,SPM)是显微镜的一个分支,它利用物理探针扫描标本形成样本表面图像.而原子力显微镜(Atomic force microscopy,AFM)是SPM中一种多功能的表面成像和测量工具,对导电、不导电、真空中、空气中或流体中的各种样本均可测量.原子力显微镜最面临的最大挑战之一是评估其在表面测量过程中所伴随的不确定度.本研究通过XYZ Phase的标定,对一台光学原子力显微镜进行了校准.该方法旨在克服在评估一些无法实验确定的不确定部件时遇到的困难,如尖端表面相互作用力和尖端几何.运用蒙特卡罗方法来确定根据相关容差和概率密度函数(PDFs)随机绘制参数而引起的相关不确定度.整个过程遵循《测量不确定度表示指南》(GUM)补编2.经本方法验证,原子力显微镜的评估不确定度为10nm左右.     

一种高精度多功能双用原子力显微镜技术及应用

主要研究了一种基于高精度IPC-205B型扫描隧道显微镜(STM)的新型高精度多功能双用原子力显微镜(AFM)技术及其应用.阐述该原子力显微镜的工作原理、组成及应用,详细介绍了该AFM镜体的独特结构和新型微悬臂的制作及其检测方法.该AFM采用简单适用的新型微悬臂.并利用STM检测微悬臂的起伏,通过四维机械驱动和双压电陶瓷扫描,有效提高了扫描精度,扩大了扫描范围.该机型集AFM和STM功能为一体,其中STM可以单独使用.该机型检测精度可达:横向0.1 nm,纵向0.01 nm.并用该样机进行了样品表面形貌和隧道谱的实验研究.     

Nanodissection and noncontact imaging of plasmid DNA with an atomic force microscope

In this paper we report that a combination of noncontact and contact atomic force microscopy is a convenient and reliable method for imaging and dissecting single plasmid deoxyribonucleic acid (DNA) strands on mica at ambient conditions without leaving feedback and without damage to the scanning tips. The width and thickness measured at different points of the DNA strands agree with literature data and are the same before and after dissection.     

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.     

大范围扫描原子力显微镜自动调平控制技术

为了进一步扩大原子力显微镜(AFM)的应用范围,研制出一套大范围高速AFM系统.该系统采用上、下两个扫描器,上扫描器负责Z方向闭环控制的动态响应,下扫描器负责X、Y方向平面扫描及Z方向补偿控制.针对样品放置倾斜对大范围扫描成像的影响,提出基于多线扫描的样品自动调平控制技术.首先通过多线扫描确定样品倾斜位置,然后将所有扫描点的倾斜位移差用函数式表达,最后将位移差换算为控制电压作为扫描器Z向的前馈控制输入.实验结果表明,能消除样品倾斜对AFM大范围扫描的影响.     

A convenient and rapid sample repositioning approach for atomic force microscopy

A novel repositioning approach is described for repeated observations of a specimen at a close proximal location in the atomic force microscope. The approach is similar to keystone architecture, whereby the repositioning is achieved by forming a male structured base for the specimen, and a corresponding female counterpart as the frame. For the combination of an acrylic acid frame and a metal base, 90% translation shifts are less than 10 µm, and almost all angular disorientations are within +3° to ?3°. Nanometre‐scale surface features can be relocated easily and reliably even after 40 imaging–removal–imaging cycles, dipping the specimen in solutions or heating up to 500 °C.     

Accurate measurement of the out-of-plane motion of a tip-scanning atomic force microscope

A tip-scanning atomic force microscope (AFM) can be used as a highly accurate height-measuring instrument for large samples, such as liquid crystal displays. To accurately measure the flatness or surface roughness of large samples, the xy-scanner-induced out-of-plane motion must be known to discriminate scanner artifacts from the measured AFM images. As the topographic signals of AFM measurements contain the hysteresis of the z-scanner piezoelectric actuators, actual movements of the z-scanner were measured using a z-axis sensor glued to the actuator. The actual out-of-plane motion of the xy-scanner was found to be less than 1 nm for a 50-μm scan.     

Comparison of friction measurements using the atomic force microscope and the surface forces apparatus: the issue of scale

Results are presented of lateral force measurements using the atomic force microscope (AFM) and the surface forces apparatus (SFA). Two different probes are used in the AFM measurements; a sharp silicon nitride tip (radius R20 nm) and a glass ball (R15 m). The lateral force is measured between the (silicon nitride or glass) probe and a mica surface which has been coated by a thin lubricant film. In the SFA, a thin lubricant film separates two molecularly smooth mica surfaces (R1 cm) which are slid relative to each other. Perfluoropolyether (PFPE) and polydimethylsiloxane (PDMS) were used as the lubricant films. In the SFA where the contact diameter is largest, the PFPE film shows much lower friction than PDMS. As the size of the probe decreases, the difference in the measured friction decreases. For sharp AFM tips, no clear distinction between the tribological properties of the films can be made. Hence, the measured coefficient of friction varies according to the length scale probed, at least for small dimensions.     

Polysaccharide properties probed with atomic force microscopy

In recent years, polysaccharides have been extensively studied using atomic force microscopy (AFM). Owing to its high lateral and vertical resolutions and ability to measure interaction forces in liquids at pico‐ or nano‐Newton level, the AFM is an excellent tool for characterizing biopolymers. The first imaging studies showed the morphology of polysaccharides, but gradually more quantitative image analysis techniques were developed as the AFM grew easier to use in aqueous liquids and in non‐contact modes. Recently, AFM has been used to stretch polysaccharides and characterize their physicochemical properties by application of appropriate polymer stretching models, using a technique called single‐molecule force spectroscopy. From application of such models as the wormlike chain, freely jointed chain, extensible‐freely jointed chain, etc., properties such as the contour length, persistence length and segment elasticity or spring constant can be calculated for polysaccharides. The adhesion between polysaccharides and surfaces has been quantified with AFM, and this application is particularly useful for studying polysaccharides on microbial and other types of cells, because their adhesion is controlled by biopolymer characteristics. This review presents a synthesis of the theory and techniques currently in use to probe the physicochemical properties of polysaccharides with AFM.     

Long-term structural changes of plasmid DNA studied by atomic force microscopy

Long-term stability of plasmid DNA (pDNA) conformations is critical in many research areas, especially those concerning future gene therapy. Despite its importance, the time-evolution of pDNA structures has rarely been studied at a molecular resolution. Here, the time-evolution of pDNA solutions spanning four years was observed with atomic force microscopy (AFM). The AFM data show that the pDNA molecules changed over time from isolated supercoiled structures, to aggregated supercoiled structures, to thin, branched network structures, and finally to wider, branched network structures. Additional topographical analysis of the AFM data suggests that the actions of residual proteins could be the main mechanism for the structural changes in our laboratory-prepared pDNA.     

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。     

Manipulation, dissection, and lithography using modified tapping mode atomic force microscope

A modified tapping mode of the atomic force microscope (AFM) was introduced for manipulation, dissection, and lithography. By sufficiently decreasing the amplitude of AFM tip in the normal tapping mode and adjusting the setpoint, the tip-sample interaction can be efficiently controlled. This modified tapping mode has some characteristics of the AFM contact mode and can be used to manipulate nanoparticles, dissect biomolecules, and make lithographs on various surfaces. This method did not need any additional equipment and it can be applied to any AFM system.     

New direct observations of asphalts and asphalt binders by scanning electron microscopy and atomic force microscopy

Observations made using AFM and SEM have been combined in order to study the structure of asphalts. Fluorescence microscopy was used to aid in understanding the structural changes occurring when polymer is added to the asphalts.   With the atomic force microscope we are able to study the structure of the asphalts without any pre-preparation. Despite very low resolution, our study reveal ed a network of asphaltene molecules with regard to asphalt gel. The same result is obtained by SEM observation but with a much better resolution. SEM observation, however, needs an adequate preparation method.   In the presence of polymer we observed a rearrangement of the initial asphaltene association which leads to the assumption that polymer can aggregate the asphaltene phase.