Fabrication of various tip-size AFM probes for evaluating single-molecular retraction force between actin and anti-actin

The authors fabricated a probe tip with various sizes and examined the size dependency of the probe tip on the distribution of retraction forces between actin and anti-actin. Probe tips of various sizes were fabricated by two-photon polymerization methods on a micro cantilever of an atomic force microscope (AFM). The authors succeeded in fabricating a spherical tip having a smooth surface and the tip size varied between φ 0.8 and 5.5 μm. Anti-actin was immobilized on the fabricated probe tips and force curves were measured against an actin-immobilized mica substrate by AFM to analyze the retraction forces. The histograms of retraction forces showed that the single-molecular retraction force between actin and anti-actin was ca. 350–400 pN. It was observed that the average retraction forces for each tip size correlated with the square of the tip radius.     

双探针原子力显微镜视觉对准系统

传统的原子力显微镜（AFM）受针尖形状和放置方式的影响很难测量线条的宽度和两个侧壁的形状,故本文提出采用双探针对顶测量方案来消除AFM针尖形状对测量结果的影响。介绍了一种基于机器视觉的双探针原子力显微镜对准系统,该系统将两个探针接触到一起,实现了双探针在三维方向上的对准。系统采用具有亚微米级分辨率的镜头,配合高分辨率的CCD来获得探针的清晰图像,用于在水平和垂直两个方向实时监控双探针的运动情况。采用基于石英音叉式的自传感自调节的原子力探针,无需外加光学探测系统,缩小了系统体积,避免了杂散光对视觉对准系统的干扰。最后对针尖进行了亚像素边缘提取,精确地获取了探针之间的相对位置,实现了亚微米级的双探针对准（1 μm以内）。该结论由探针之间距离与幅度/相位曲线得到了验证。     

基于AFM的化学机械抛光磨粒模拟研究

当前的化学机械抛光(CMP)磨损模型中大多数都缺少微观试验数据的支持。为进步揭示CMP中纳米磨粒对材料表面的磨损机制,提出了采用原子力显微镜(AFM)来模拟CMP中的单个磨粒的试验方案,并验证该模拟试验方案的可行性。结果表明:采用AFM探针来模拟CMP过程中单个磨粒对芯片表面的磨损与相互作用的试验方案是完全可行的;可以通过AFM探针对芯片表面的相互作用与磨损,模拟得出单个磨粒对芯片表面的作用与磨损状况,并可以在此微观试验数据的基础上建立起新的CMP磨损模型。     

Analysis of force curves obtained on the live cell membrane using chemically modified AFM probes

Force curves were obtained on the live cell surface using an atomic force microscope mounted with a modified tip with the bifunctional covalent crosslinker, disuccinimidyl suberate, which forms a covalent bond with amino-bearing molecules on the cell surface. A ramp delay time of 1.0 s was introduced before the start of the retraction regime of the force curve to increase the stationary reaction time between the crosslinkers on the tip and the amino groups on the cell surface. While live cell surface responses to forced contact with a non-functionalized tip rarely showed evidence of tip–cell interaction, those obtained with modified tips gave clear indication of prolonged adhesion which was terminated by a single step release of the tip to its neutral position. Under the given experimental conditions of this work, 58% of a total of 198 force curves gave only one jump and 70% of those with one jump gave the final rupture force of 4.5±0.22 nN. The result emphasized the uniqueness of the observed mechanical response of the cell surface when probed with chemically modified tips.     

A novel PRC signal drift reduction method for new developed SEM-based nanoindentation/nanoscratch instrument integrated with AFM

In-situ SEM nanoindentation and nanoscratch testing methods are commonly used for mechanical characterization and investigation of the deformation and failure mechanisms of coating materials with micro-to nano-scale thicknesses. However, existing SEM-based integrated nanoindentation and nanoscratch instruments have two main limitations. First, the measured mechanical properties of the coating materials at micro-to nano-scale thicknesses are highly sensitive to surface roughness. Second, the existing SEM-based instruments lack the capability to acquire the morphology of residual imprints in real-time after nanoindentation and nanoscratching. In this study, a novel SEM-based integrated nanoindentation, nanoscratch, and atomic force microscopy (AFM) instrument, namely, NMT-AFM was proposed, developed and fabricated. The self-sensing piezoresistive cantilever (PRC) was selected as the AFM force sensor owing to its miniaturization ability. However, the resistance of the PRC sensor fluctuated because of the electron irradiation from SEM, resulting in the continuous drift of the PRC signal during SEM imaging. To overcome this limitation, a mechanism of PRC signal drift inside SEM was analyzed for the first time, and a PRC signal drift reduction method was proposed based on the mechanism analysis. The experimental results indicated that the PRC signal drift was reduced to 2 nm in 2 min by applied external voltage value U_A of 30 V to modified PRC, which proved the proposed mechanism of PRC signal drift during SEM imaging. Finally, the X–Y fine nanopositioner angle calibration test using AFM calibration chip VGRP-UM and the nanoindentation/nanoscratch characterizations of the TiAlSiN coating material were conducted.     

Study of the sensitivity and resonant frequency of the torsional modes of an AFM cantilever with a sidewall probe based on a nonlocal elasticity theory

A relationship based on a nonlocal elasticity theory is developed to investigate the torsional sensitivity and resonant frequency of an atomic force microscope (AFM) with assembled cantilever probe (ACP). This ACP comprises a horizontal cantilever and a vertical extension, and a tip located at the free end of the extension, which makes the AFM capable of topography at sidewalls of microstructures. First, the governing differential equations of motion and boundary conditions for dynamic analysis are obtained by a combination of the basic equations of nonlocal elasticity theory and Hamilton's principle. Afterward, a closed‐form expression for the sensitivity of vibration modes has been obtained using the relationship between the resonant frequency and contact stiffness of cantilever and sample. These analysis accounts for a better representation of the torsional behavior of an AFM with sidewall probe where the small‐scale effect are significant. The results of the proposed model are compared with those of classical beam theory. The results show that the sensitivities and resonant frequencies of ACP predicted by the nonlocal elasticity theory are smaller than those obtained by the classical beam theory. Microsc. Res. Tech. 78:408–415, 2015. © 2015 Wiley Periodicals, Inc.     

High-resolution constant-height imaging with apertured silicon cantilever probes

We present high-resolution aperture probes based on non-contact silicon atomic force microscopy (AFM) cantilevers for simultaneous AFM and near-infrared scanning near-field optical microscopy (SNOM). For use in near-field optical microscopy, conventional AFM cantilevers are modified by covering their tip side with an opaque aluminium layer. To fabricate an aperture, this metal layer is opened at the end of the polyhedral probe using focused ion beams (FIB). Here we show that apertures of less than 50 nm can be obtained using this technique, which actually yield a resolution of about 50 nm, corresponding to λ/20 at the wavelength used. To exclude artefacts induced by distance control, we work in constant-height mode. Our attention is particularly focused on the distance dependence of resolution and to the influence of slight cantilever bending on the optical images when scanning at such low scan heights, where first small attractive forces exerted on the cantilever become detectable.     

Mapping of enzyme activity by detection of enzymatic products during AFM imaging with integrated SECM-AFM probes

With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy (SECM) with AFM has recently been introduced. Simultaneous contact mode imaging of a micropatterned sample with immobilized enzyme spots and imaging of enzyme activity is shown. In contrast to force spectroscopy the conversion of an enzymatic byproduct is directly detected during AFM imaging and correlated to the activity of the enzyme.     

Fabrication of carbon nanotube AFM probes using the Langmuir–Blodgett technique

Carbon nanotube (CNT)-tipped atomic force microscopy (AFM) probes have shown a significant potential for obtaining high-resolution imaging of nanostructure and biological materials. In this paper, we report a simple method to fabricate single-walled carbon nanotube (SWNT) nanoprobes for AFM using the Langmuir–Blodgett (LB) technique. Thiophenyl-modified SWNTs (SWNT-SHs) through amidation of SWNTs in chloroform allowed to be spread and form a stable Langmuir monolayer at the water/air interface. A simple two-step transfer process was used: (1) dipping conventional AFM probes into the Langmuir monolayer and (2) lifting the probes from the water surface. This results in the attachment of SWNTs onto the tips of AFM nanoprobes. We found that the SWNTs assembled on the nanoprobes were well-oriented and robust enough to maintain their shape and direction even after successive scans. AFM measurements of a nano-porous alumina substrate and deoxyribonucleic acid using SWNT-modified nanoprobes revealed that the curvature diameter of the nanoprobes was less than 3 nm and a fine resolution was obtained than that from conventional AFM probes. We also demonstrate that the LB method is a scalable process capable of simultaneously fabricating a large number of SWNT-modified nanoprobes.     

Quantitative measurement of mRNA at different loci within an individual living cell

Asymmetric localizations of cellular proteins and mRNAs are important for cell functions such as division, differentiation and development. The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins and thereby restricting their locations to appropriate cellular regions. We have previously reported a novel method based on atomic force microscopy (AFM) for examining gene expression in a single living cell without killing or destroying it. An AFM tip was inserted into a living cell to extract mRNAs, which were analyzed after multiplication by RT-PCR and quantitative PCR. By applying this method, in this study we performed quantitative measurement of mRNA at different loci within individual living cells.     

Dynamic spring constants for higher flexural modes of cantilever plates with applications to atomic force microscopy

In atomic force microscopy (AFM) a sharp tip fixed close to the free end of a cantilever beam interacts with a surface. The interaction can be described by a point-mass model of an equivalent oscillator with a single spring located at the position of the tip. However, other spring constants have to be used to describe the oscillation behavior correctly if forces are acting on the cantilever over an extended lateral range. A point-mass model is then no longer valid. In the present study we derive expressions for the spring constants of cantilevers that can interact with any part of their plan view area along the beam and for all flexural modes. The equations describe the oscillation behavior in the corresponding mass model and are based on the eigenfrequencies and modal shapes of the free cantilever. The results are of high practical relevance, for example if an AFM is operated in a higher flexural mode, if the tip is not located at the free end of the cantilever beam, or if the external conservative forces affecting cantilever movement are not restricted to a single point. The limitations of the approach are discussed.     

Influence of the tip mass and position on the AFM cantilever dynamics: Coupling between bending,torsion and flexural modes

The effects of the geometrical asymmetric related to tip position as a concentrated mass, on the sensitivity of all three vibration modes, lateral excitation (LE), torsional resonance (TR) and vertical excitation (VE), of an atomic force microscopy (AFM) microcantilever have been analyzed. The effects of the tip mass and its position are studied to report the novel results to estimating the vibration behavior of AFM such as resonance frequency and amplitude of the microcantilever. In this way, to achieve more accurate results, the coupled motion in all three modes is considered. In particular, it is investigated that performing the coupled motion in analysis of AFM microcantilever is almost necessary. It is shown that the tip mass and its position have significant effects on vibrational responses. The results show that considering the tip mass decreases the resonance frequencies particularly on high-order modes. However, dislocating of tip position has an inverse effect that causes an increase in the resonance frequencies. In addition, it has been shown that the amplitude of the AFM microcantilever is affected by the influences of tip and its position. These effects are caused by the interaction between flexural and torsional motion due to the moment of inertia of the tip mass.     

Morphological, nanomechanical and cellular structural characterization of human hair and conditioner distribution using torsional resonance mode with an atomic force microscope

Characterization of the cellular structure and chemical and physical properties of hair are essential to develop better cosmetic products and advance the biological and cosmetic sciences. Although the morphology of the fine cellular structure of human hair has traditionally been investigated using scanning electron microscopy and transmission electron microscopy, atomic force microscopy can be used for characterization in ambient conditions without requiring specific sample preparations and surface treatment. In this study, the tapping and torsional resonance modes in an atomic force microscope are compared for measurements of stiffness and viscoelastic properties. The materials were mapped using amplitude and phase angle imaging. The torsional resonance mode showed advantages in resolving the in-plane (lateral) heterogeneity of materials. This mode was used for investigating and characterizing the fine cellular structure of human hair. Various cellular structures (such as the cortex and the cuticle) of human hair and fine sublamellar structures of the cuticle, such as the A-layer, the exocuticle, the endocuticle and the cell membrane complex were easily identified. The distribution and thickness of conditioner on the treated hair surface affects the tribological properties of hair. The thickness of the conditioner was estimated using force distance measurements with an atomic force microscope.     

Comparisons of the topographic characteristics and electrical charge distributions among Babesia‐infected erythrocytes and extraerythrocytic merozoites using AFM

Tick‐borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite–host relationships. We hereby used atomic force microscopy (AFM) and frequency‐modulated Kelvin probe potential microscopy (FM‐KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM‐KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis‐infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi‐infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium‐infected erythrocytes, FM‐KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis‐infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.     

Study of the nanoscopic deformation of an annealed nafion film by using atomic force microscopy and a patterned substrate

We demonstrated the repetitive imaging of the same area of a nafion film before and after annealing by using atomic force microscopy (AFM). In order to find the exact same area of the same sample after changing the cantilever and reattaching the sample, a micropatterned substrate was developed. A micropattern with a 250–500 μm pitch was prepared on the backside of a transparent glass substrate. This pattern includes various signs such as colored letters and numbers at the center of each lattice of the pattern. The nanostructures fabricated by AFM nanolithography on a nafion film using this new method were successfully characterized before and after annealing (over 100 °C). The AFM images clearly showed that the nanostructures on a nafion film were dramatically changed by annealing. The data indicated an evidence to understand why the nafion fuel cell does not work well at high temperatures. Our method is probably effective for the study of nanoscopic dynamics in various surface structures.     

T细胞与K562细胞共培养的AFM观察

人类机体的免疫系统很难彻底清除肿瘤细胞,了解T淋巴细胞杀伤肿瘤细胞的整个过程的机理,将为提高免疫系统杀灭肿瘤的效率提供知识基础。本文采用原子力显微镜与倒置显微镜在细胞层面上观察T淋巴细胞进攻杀伤人慢性粒细胞性白血病细胞株——K562细胞的过程,并对T细胞与K562细胞共培养前后的表面形貌和生物机械性质进行表征。结果显示,与培养前相比,共培养后2种细胞数目之和减少,2种细胞的表面形貌和机械力学性质均出现很大差异,表现为:K562细胞,平均粗糙度(Ra)降低,细胞平均高度(Mh)降低,细胞表面出现5～8 cm的孔洞,有的细胞甚至完全破裂溶解。多个T细胞的统计分析结果显示,共培养前,T细胞为静息状态,而共培养后Ra和Mh都显著增加。该方法为研究免疫系统与肿瘤相互作用的机制提供重要的切入点。     

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.     

Bridging the gap between conventional and video-speed scanning probe microscopes

A major disadvantage of scanning probe microscopy is the slow speed of image acquisition, typically less than one image per minute. This paper describes three techniques that can be used to increase the speed of a conventional scanning probe microscope by greater than one hundred times. This is achieved by the combination of high-speed vertical positioning, sinusoidal scanning, and high-speed image acquisition. These techniques are simple, low-cost, and can be applied to many conventional microscopes without significant modification. Experimental results demonstrate an increased scan rate from 1 to 200 Hz. This reduces the acquisition time for a 200×200 resolution image from 3 min to 1 s.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.