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.     

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.     

Dual frequency atomic force microscopy on charged surfaces

The cantilever is mechanically driven at two resonant frequencies in a bimodal atomic force microscope (AFM). To generate the feedback signal for topography measurement the deflection signal is demodulated at one frequency and for compositional surface mapping at the other. In particular, the second mode amplitude and phase signals are used to map surface forces such as the van der Waals interaction. On electrically charged surfaces both, van der Waals forces and electrostatic forces contribute to the second eigenmode signal. The higher eigenmode signal in bimodal AFM reflects the local distribution of electrical charges. Mechanically driven bimodal AFM thus also provides a valuable tool for compositional mapping based on surface charges.     

Near-grain-boundary characterization by atomic force microscopy

Characterization of near-grain boundary is carried out by atomic force microscopy (AFM). It has been observed to be the most suitable technique owing to its capability to investigate the surface at high resolution. Commercial purity-grade nickel processed under different conditions, viz., (i) cold-rolled and annealed and (ii) thermally etched condition without cold rolling, is considered in the present study. AFM crystallographic data match well with the standard data. Hence, it establishes two grain-boundary relations viz., plane matching and coincidence site lattice (CSL Σ=9) relation for the two different sample conditions.     

AFM纳米加工系统设计

利用自行研制原子力显微镜开发了一套纳米加工系统,系统利用软件预先读入图形,根据图形控制探针的精确走向,实现复杂图案的矢量式扫描刻蚀,并且对陶瓷管的非线性畸变实现了软件矫正,实验表明系统可加工出精细的纳米图形,为进一步加工出更为精细复杂的纳米器件奠定了基础。     

Method of imaging low density lipoproteins by atomic force microscopy

This short paper reports a simple method to image low density lipoproteins (LDL) using atomic force microscopy (AFM). This instrument allows imaging of biological samples in liquid and presents the advantage of needing no sample preparation such as staining or fixation that may affect their general structure. Dimensions (diameter and height) of individual LDL particles were successfully measured. AFM imaging revealed that LDL have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in the z axis (i.e., height). LDLs were found to have an average diameter of 23 +/- 3 nm. The obtained mean height was 10 +/- 2 nm.     

Micropatterning of bacteria on two-dimensional lattice protein surface observed by atomic force microscopy

In this study, we characterized the two-dimensional lattice of bovine serum albumin (BSA) as a chemical and physical barrier against bacterial adhesion, using fluorescence microscopy and atomic force microscopy (AFM). The lattice of BSA on glass surface was fabricated by micro-contact printing (muCP), which is a useful way to pattern a wide range of molecules into microscale features on different types of substrates. The contact-mode AFM measurements showed that the average height of the printed BSA monolayer was 5-6nm. Escherichia coli adhered rapidly on bare glass slide, while the bacterial adhesion was minimized on the lattices in the range of 1-3mum(2). Especially, the bacterial adhesion was completely inhibited on a 1mum(2) lattice. The results suggest that the anti-adhesion effects are due by the steric repulsion forces exerted by BSA.     

Effect of cantilevers' dimensions on phase contrast in multifrequency atomic force microscopy

During the past years, different theoretical and experimental works are done to enhance the observables (mostly higher eigenmode's phase contrast) in multifrequency atomic force microscopy methods. In this study, the geometry of rectangular cantilevers is studied and an optimum dimension that can provide maximum phase contrast for a given set of samples is found. The analysis is done both numerically and experimentally. A sensitivity analysis is provided to demonstrate which dimension (length, width, thickness, tip‐radius, and cantilever and sample angle) of the cantilever has a higher effect on the results. The effects of geometrical dimensions are categorized into to: (a) effect on dynamics of the cantilever (b) effects on cantilever's specifications (i.e., spring constant and quality factor). Length and width of the cantilever dominates the static behavior of the cantilever. While thickness (for lower values), tip radius, and approach angle mostly affect the dynamic behavior of the cantilever. Theoretically, it is found as the length increases the phase contrast increase. This relationship is opposite for width. It was also observed that the effect of thickness for a specific range on the phase contrast depends on the 1st eigenmode amplitude setpoint. This study shows for having higher contrast, lower tip‐radius is needed. The optimum angle between cantilever and sample to enhance bimodal atomic force microscopy imaging is also found. Based on the commercially available cantilevers, the optimum cantilever dimension is provided. Three different cantilevers with similar dimensions are experimentally tested and theoretical results are verified.     

Mechanical properties of asphalt binders evaluated by atomic force microscopy

Atomic Force Microscopy was employed in order to relate the features observed on the surface of a 50/70 asphalt binder according to its local stiffness and elastic recovery. Indentations were performed in different points of the surface and a significant variation of elasticity was observed between the points on the so-called bee structure and the matrix. Also, indentations varying the maximum force were performed on similar white spots in the bee structure and the recovery was followed up to 1 h after indentation. It was observed that the elastic recovery is very much dependent on the colloidal structure of the bee. The final surface state of the binder, close to the bee for usual bees is not the same as the initial one indicating severe plastic deformation. Also, permanent phase change could be observed for bright spots presented in not well-structured bee arrangements. A surface hardening was observed in the bee region.     

Scan speed limit in atomic force microscopy

The scan speed limit of atomic force microscopes has been calculated. It is determined by the spring constant of the cantilever k, its effective mass m, the damping constant D of the cantilever in the surrounding medium and the stiffness of the sample. Techniques to measure k, k/m and D/m are described. In liquids the damping constant and the effective mass of the cantilever increase. A consequence of this is that the transfer function always depends on the scan speed when imaging in liquids. The practical scan speed limit for atomic resolution in vacuum is 0·1 μm/s while in water it increases to about 2 μm/s due to the additional damping of cantilever movements. Sample stiffness or damping of cantilever movements by the sample increase these limits. For soft biological materials imaged in water at a desired resolution of 1 nm the scan speed should not exceed 2 μm/s.     

Intrinsic dissipation in atomic force microscopy cantilevers

In this paper we build a practical modification to the standard Euler-Bernoulli equation for flexural modes of cantilever vibrations most relevant for operation of AFM in high vacuum conditions. This is done by the study of a new internal dissipation term into the Euler-Bernoulli equation. This term remains valid in ultra-high vacuum, and becomes particularly relevant when viscous dissipation with the fluid environment becomes negligible. We derive a compact explicit equation for the quality factor versus pressure for all the flexural modes. This expression is used to compare with corresponding extant high vacuum experiments. We demonstrate that a single internal dissipation parameter and a single viscosity parameter provide enough information to reproduce the first three experimental flexural resonances at all pressures. The new term introduced here has a mesoscopic origin in the relative motion between adjacent layers in the cantilever.     

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.     

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.     

The effect of deformation on the lateral resolution of atomic force microscopy

A computer model based on the elastic properties of rubber is introduced for the evaluation of the lateral resolution in atomic force microscopy of deformable specimens. The computational results show that, if the full width at half-height can be defined as the lateral resolution, it is continuously improved at greater probe forces, at the expense of a reduced molecular height. In fact, even for a probe that is bigger than the molecule, the real size of the molecule can be 'recovered' at about 25% compression. This result demonstrates that for a better lateral resolution, a greater probe force can be beneficial, provided that the molecule is not moved or damaged and the response remains elastic. Measurements on isolated low-density lipoproteins (LDL) show that with 26% vertical compression, the lateral size measured in atomic force microscopy is only about 72% of the value predicted by a simple convolution, and is only slightly larger (≈ 13%) than the known size of LDL. Therefore, the results on LDL provide a direct support for the conclusions of the computational model.     

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.     

Visualization of cellular DNA crosslinks by atomic force microscopy

Cellular DNA crosslinks are a type of DNA damage induced by toxic chemicals or high‐energy radiation. If damaged DNA is not rapidly repaired, cells will die or mutate. To evaluate the types of DNA damage and their influence on vital cell activities, it is necessary to be able to detect DNA crosslinks. To date, indirect methods such as alkaline elution, potassium chloride–sodium dodecyl sulfate assay and comet assay have been used to detect DNA damage. Direct morphological observation, on the other hand, may be a useful tool to differentiate the types of DNA damage. In this report, atomic force microscopy (AFM) has been employed to visualize the breakage and crosslinking of cellular DNA strands in cells treated with formaldehyde and hydrogen peroxide. Our results showed that toxic chemical‐induced crosslinking of cellular DNA occurred in a dose‐dependent manner. DNA conglomerates were observed with high concentrations of formaldehyde, and the AFM observations were consistent with those of a comet assay. Our experiments demonstrate that AFM is an efficient method to differentiate the types of DNA damage. SCANNING 31: 75–82, 2009. © 2009 Wiley Periodicals, Inc.     

High-resolution imaging of chromosome-related structures by atomic force microscopy

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein.     

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.     

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.     

Detection of erythrocytes in patients with multiple myeloma using atomic force microscopy

The goal of this study was to examine the pathophysiological changes to erythrocytes in multiple myeloma (MM) patients at a nanometer scale. We hypothesize that studying changes in red blood cells may be important for early diagnosis and effective treatment of MM. Blood samples were taken from ten healthy volunteers and ten MM patients before and after treatment. Changes in the morphological and biomechanical properties of the erythrocytes were studied at a nanometer scale with atomic force microscopy (AFM). There were dramatic deformations in the overall shape and surface membrane of the erythrocytes from pre‐ and post therapeutic MM patients compared with the healthy controls. Healthy and pathological MM erythrocytes could be distinguished by several morphologic parameters, including the width, length, length to width ratio, valley, peak, valley‐to‐peak, standard deviation, and surface fluctuation. The effectiveness of disease treatment could also be evaluated by studying these red blood cell parameters. AFM was able to detect noticeable morphological differences in the red blood cells from MM patients compared with healthy controls. Therefore, erythrocyte morphology is an important parameter for diagnosing MM, as well as evaluating the efficacy of disease treatment. SCANNING 34: 295–301, 2012. © 2012 Wiley Periodicals, Inc.