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.     

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.     

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.     

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.     

Construction of pcAFM module to measure photoconductance with a nanoscale spatial resolution

A photoconductive atomic force microscopy (pcAFM) module was designed and the performance was tested. This module consisted of three units: the conductive mirror-plate, the steering mirror and the laser source. The module with a laser irradiation unit was equipped to a conventional conducting probe atomic force microscopy (CP-AFM) instrument to measure photoconductance in a nanoscale resolution. As a proof-of-concept experiment, the photoconductance of aggregated fullerene on indium tin oxide (ITO) substrate was measured with this module. The electrical signals (currents) of aggregated fullerene under the conditions of laser on/off at about -10V sample bias voltage were -100 to -160nA and 0 to -20nA, respectively. Results indicated that the pcAFM with this module allowed one to observe photoinduced changes of electrical properties in nanodevices with nanoscale spatial resolution.     

Contact mechanics and tip shape in AFM-based nanomechanical measurements

Stiffness-load curves obtained in quantitative atomic force acoustic microscopy (AFAM) measurements depend on both the elastic properties of the sample and the geometry of the atomic force microscope (AFM) tip. The geometry of silicon AFM tips changes when used in contact mode, affecting measurement accuracy. To study the influence of tip geometry, we subjected ten AFM tips to the same series of AFAM measurements. Changes in tip shape were observed in the scanning electron microscope (SEM) between individual AFAM tests. Because all of the AFAM measurements were performed on the same sample, variations in AFAM stiffness-load curves were attributed to differences in tip geometry. Contact-mechanics models that assumed simple tip geometries were used to analyze the AFAM data, but the calculated values for tip dimensions did not agree with those provided by SEM images. Therefore, we used a power-law approach that allows for a nonspherical tip geometry. We found that after several AFAM measurements, the geometry of the tips at the very end is intermediate between those of a flat punch and a hemisphere. These results indicate that the nanoscale tip-sample contact cannot easily be described in terms of simple, ideal geometries.     

Probe three-dimensional structure of soft organized surfactants at solid–liquid interface with an AFM in MAC Mode and contact mode

Atomic force microscopy provides a unique direct-visualization tool to study the three-dimensional structure of adsorbed surfactants on solid surfaces. Ionic surfactant molecules spontaneously adsorbed onto hydrophilic surfaces from aqueous solution above the critical micelle concentration (cmc) have been imaged using an atomic force microscope in magnetic ac mode (MAC Mode) and contact mode. It was found that the soft organized surfactants were highly compressible and therefore showed a wide range of corrugations depending on imaging forces. When using gentle MAC Mode, corrugations of the organized surfactants around half of the estimated height of the proposed surfactant aggregate cylinders on mica have been stably observed. Traditional contact mode operating in the pre-contact double-layer electrostatic interaction region, however, showed significantly reduced height of the organized molecules.     

Fabrication of hierarchical micro/nanostructures via scanning probe lithography and wet chemical etching

In this study, we propose a simple and effective method for fabricating hierarchical silicon structures via the combination of scanning probe lithography (SPL) and wet chemical etching. Here, silicon oxide structures were protruded from a 100-oriented silicon surface, followed by the passivation of silicon nitride by AFM tip-induced local oxidation. Based on the two-dimensional (2D) silicon oxide patterns, three-dimensional (3D) microstructures with high aspect ratios were formed by wet etching with HF and KOH. A variety of combinations of SPL and the etching process allowed us to fabricate diverse silicon-based structures such as deep-etched microstructures and multi-terraced nanostructures.     

Optimization of the film coating process for polymer blends by the grey-based Taguchi method

The purpose of this paper is to find the optimum conditions of the film coating process for polymer blends and the influence of major processing parameters on the morphological properties of the material surface has been discussed. The surface roughness is regarded as the target characteristic of the smaller-the-better system. In order to achieve the aim of the multiple-response process of robustness, the grey-based Taguchi method is proposed. Nine experimental trials based on the L₉(3⁴) orthogonal array are conducted to determine the optimum processing conditions, the significant factor levels, and the percent contributions together with the analysis of variance (ANOVA). Also, confirmation experiments are performed to verify that the experimental results are reproducible. Moreover, atomic force microscopy (AFM) can be carried out with 3D atomic resolution in air for the measurement and analysis of the precision surface .     

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.     

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.