Prototype cantilevers for quantitative lateral force microscopy

Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The "hammerhead" cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever "torque sensitivity" to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.     

Combination of AFM with an objective-type total internal reflection fluorescence microscope (TIRFM) for nanomanipulation of single cells

A new instrument was constructed by combining an objective-type total internal reflection fluorescence microscope with an atomic force microscope (AFM). Our purpose of constructing such an instrument is to detect and confirm the result of cellular level manipulations made with the AFM part through the detection system of the highly sensitive fluorescence microscope part. In this combination, manipulations are now possible from the nanometer to the micrometer scales and the fluorescence detection system is sensitive enough even for localizing single molecules. In this paper, we applied the system as a precise intracellular injector (nanoplanter). Fluorescent beads were first chemically immobilized onto a ZnO whisker that was glued to an AFM tip and were injected into a living BALB/3T3 cell together with the whisker. It was demonstrated that the system could clearly show the result of injection, that is, the presence of a small number of fluorescent beads in the cell.     

Imaging DNA molecules on mica surface by atomic force microscopy in air and in liquid

DNA molecules immobilized on mica surface by various methods have been observed by atomic force microscopy both in air and in liquid. Divalent cations and 3-aminopropyltriethoxysilane (APTES) modified mica surface have been used to immobilize the DNA molecules. Optimal DNA and divalent cations concentration for AFM imaging are presented. Among the different methods of modifying mica surface with APTES, the water solution modifying method appears to get the best results. When using high DNA concentration for AFM imaging, DNA networks can be formed. A simple method to extend long DNA molecules is demonstrated. The optimal imaging conditions and AFM operating techniques are discussed. Different DNA immobilizing methods have been compared and evaluated.     

Atomic force microscopy characterization of the chemical contrast of nanoscale patterns fabricated by electron beam lithography on polyethylene glycol oxide thin films

The present paper shows that atomic force microscopy (AFM) imaging of friction force and phase lag in ambient air can be used to characterize the chemical contrast induced by electron beam (EB) irradiation on polyethylene glycol oxide (PEO) surface. Time-of-flight secondary emission mass spectroscopy measurements showed that the EB irradiation generates chemical contrast on PEO surface by decreasing the ether bond density. The AFM measurements showed smaller phase lag and lower friction and adhesive forces on the EB irradiated PEO surface, as compared to the non-irradiated PEO surface. While the chemical contrast in friction force had a linear dependence on the EB irradiation dose, the dependence of the chemical contrast in the phase lag was strongly non-linear. As the friction and adhesive forces depended on the AFM probe hydrophilicity and air humidity, the contrast in friction and adhesive forces is ascribed to different capillary condensation of ambient water vapour at the AFM tip contact with the EB irradiated and non-irradiated PEO surfaces, respectively.     

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.     

Immobilization of DNA on 11-mercaptoundecanoic acid-modified gold (111) surface for atomic force microscopy imaging

Immobilized DNA on preformed 11-mercaptoundecanoic acids (MUDA) self-assembled monolayers (SAMs) on a gold (111) surface was bound by a divalent cation bridges was imaged by atomic force microscopy (AFM). The DNA immobilization was attributed to the formation of ionic bridges between the carboxylate groups of MUDA and the phosphate groups of DNA. AFM images revealed that DNA molecules could be immobilized strongly enough to permit stable and reproducible imaging. The effect of different bridge cations, such as Mg(2+), Zn(2+) and Cu(2+), and the pH of DNA assembled solution on immobilization and conformation of DNA was studied. Plasmid DNA pBR 322/Pst I molecules were straightened by using a molecular combing technique on the MUDA surface.     

Development of a microlateral force sensor and its evaluation using lateral force microscopy

A microlateral force sensor (MLFS) was developed and evaluated using atomic force microscopy (AFM). The sensor was attached to a sensing table supported by a suspension system. The lateral motion of the sensing table was activated by a comb actuator. The driving voltage to the comb actuator was controlled to maintain a constant position of the sensing table by detecting the tunneling current at a detector, which consisted of two electrodes where the bias voltage was applied. An AFM was used to apply a lateral force to the sensing table of the sensor. When the probe of a cantilever was pressed against the sensing table and a raster scanning was conducted, the driving voltage of the comb actuator changed to compensate the friction force between the probe and sensing table. AFM measurements of an asperity array on the sensing table were conducted, and a lateral force microscopy image (LFM) was obtained from the change in driving voltage. The image by MLFS was very similar to the LFM image that was conventionally obtained from torsion of the cantilever. The LFM image strongly correlated with the gradient image calculated from the AFM topographic image. The force sensitivity of the MLFS was determined by comparing the LFM image obtained by using the MLFS with the tangential force derived from the gradient of the AFM image.     

人脐静脉内皮细胞形貌与表面粘附力的原子力显微镜表征

目的:探讨原子力显微镜(AFM)在研究人脐静脉内皮细胞(ECV304)表面形貌、超微结构及纳米机械性质等方面的应用,讨论ECV304超微结构和机械性质与其功能的关系。方法:利用AFM对ECV304细胞的表面形貌及生物机械性质进行表征与测量。结果:在AFM下观察到用普通光学显微镜难以观察到的ECV304细胞的独特的形态结构,如细胞骨架、伪足及细胞边缘微丝等。ECV304细胞呈现长梭形、多角形、圆形等多种形态,细胞表面平均粗糙度为320.52±75.98 nm,表面均匀分布微绒毛,细胞周围有铺展的圆盘状物质。力曲线定量分析得出针尖与细胞表面的非特异性粘附力为75±14 pN。结论:通过AFM成像和力曲线测量表明,ECV304细胞呈圆形,多角形,梭形等多种形态,针尖与细胞膜表面问的粘附力比较小,约75±14pN。     

利用原子力显微镜探测化学基团间的单键力

原子力显微镜(AFM)不仅可用于形貌测量,而且还可在纳米尺度上测量微观组分间的相互作用力。本文简要介绍了AFM的一种令人非常感兴趣的用途——测量和推算单个化学基团对之间的作用力(单键力)。单键力的测量和推算涉及AFM针尖的自组装单分子膜的化学修饰和Poisson统计方法的利用,文中概述了测量和推算的步骤和原理。AFM应用范围的拓宽必将促进它的进一步改进和发展。     

Jumping mode AFM imaging of biomolecules in the repulsive electrical double layer

We present a method to image single biomolecules in aqueous media by atomic force microscope (AFM) without establishing any mechanical contact between the tip and the sample. It works by placing the feedback set point in the repulsive electrical double-layer curve just before the mechanical instability occurs. We use the jumping operation mode, where the set point is controlled at every image point and a stable imaging is achieved for several hours. This is a necessary condition for this method to be operative, otherwise the tip can fall in contact in a short time. The method is applied to image single-avidin protein molecules deposited on cleaved mica. In addition, the dependence of the height of avidin molecules as a function of ion concentration, due to differences in surface charge density of mica and avidin, is tentatively used to deduce relative values of these quantities.     

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.     

用原子力显微镜观察水流处理后的DNA图案

原子力显微镜(Atomic force microscopy,AFM)由于自身的优势,在生物领域内应用越来越广泛。同时,DNA分子由于其稳定的物理化学性质而成为纳米领域的重要实验材料,近阶段把它作为模板应用在构建纳米线等方面的研究越来越多,而怎样建立有规则图样的DNA模板就成为一个关键问题。本文介绍用原子力显微镜观察液流操纵后的DNA规则图案。     

Surface chemistry-mechanical property relationship of low density polyethylene: an IR+visible sum frequency generation spectroscopy and atomic force microscopy study

Surface-specific IR+visible sum frequency generation (SFG) spectroscopy was used to obtain chemical composition of two polymer surfaces. The SFG surface vibrational spectrum of pure low density polyethylene and that of a commercial sample of the same kind of polymer, which contains additives, are markedly different. This correlates well with the very different surface mechanical properties, i.e., stiffness (indicative of the elastic modulus) and friction, which were measured by atomic force microscopy (AFM) on the same polymer surfaces. The surface of CLDPE is dominated by methoxy (−OCH₃) contained additives, segregated from the bulk, which explains a lower stiffness, adhesion and friction of the surface, as measured by AFM. This revised version was published online in August 2006 with corrections to the Cover Date.     

Application of evanescent wave optics to the determination of absolute distance in surface force measurements using the atomic force microscope

A combined scanning near field optical/atomic force microscope (AFM) is used to obtain surface force measurements between a near field sensing tip and a tapered optical fibre surface, whilst simultaneously detecting the intensity of the evanescent field emanating from the fibre. The tapered optical fibre acts as a compliant sample to demonstrate the possible use of the near field intensity measurement system in determining 'real' surface separations from normal AFM surface force measurements at sub-nanometer resolution between deformable surfaces.     

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.     

Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.     

Fabrication of sharp tungsten-coated tip for atomic force microscopy by ion-beam sputter deposition

Tungsten (W) is significantly suitable as a tip material for atomic force microscopy (AFM) because its high mechanical stiffness enables the stable detection of tip-sample interaction forces. We have developed W sputter-coating equipment to compensate the drawbacks of conventional Si cantilever tips used in AFM measurements. By employing an ion gun commonly used for sputter cleaning of a cantilever tip, the equipment is capable of depositing conductive W films in the preparation chamber of a general ultrahigh vacuum (UHV)-AFM system without the need for an additional chamber or transfer system. This enables W coating of a cantilever tip immediately after sputter cleaning of the tip apex and just before the use in AFM observations. The W film consists of grain structures, which prevent tip dulling and provide sharpness (<3 nm in radius of curvature at the apex) comparable to that of the original Si tip apex. We demonstrate that in non-contact (NC)-AFM measurement, a W-coated Si tip can clearly resolve the atomic structures of a Ge(001) surface without any artifacts, indicating that, as a force sensor, the fabricated W-coated Si tip is superior to a bare Si tip.     

Immobilization of proteins on self-assembled monolayers

The immobilization of protein molecules on self-assembled monolayers (SAM) by physical interactions and chemical bonding has been studied using atomic force microscopy (AFM). The proteins used for our investigation are bovine serum albumin (BSA), lysozyme (LYZ), and normal rabbit immunoglobulin G (IgG). The surfaces are methyl-, hydroxyl-, carboxylic acid- and aldehyde-terminated SAMs. We found that BSA and LYZ can be readily immobilized on SAMs at their isoelectric point (IEP). The detailed surface morphology of adsorbed proteins varies with the functionality of the SAMs. The strong hydrophobic interaction at the IEP is attributed to immobilization. If the solution pH is deviated from the IEP, proteins may be attached onto the surface via electrostatic interactions. Covalent binding between the aldehyde-terminated SAM and the H2N-groups in the protein results in immobilization of all three proteins. The individual proteins and their orientations on SAMs are clearly resolved from high-resolution AFM images. The stability and bioactivity of these immobilized proteins are also studied.     

Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell

Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM). First, the distribution of the integrin beta(5) subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell. To visualize the distribution of the receptor on a living cell by an independent and potentially a more quantitative method, the AFM was used with a microbead attached to the cantilever tip to increase the area of contact and VN was immobilized on the microbead. Force measurements were then performed over a large area of a living murine osteoblastic cell using the microbead covered with VN.     

Imaging of RNA in situ hybridization by atomic force microscopy

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase-labelled antibody and visualized with 3,3'-diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate-buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock-hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA– in situ hybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before dehydration.