MAC mode atomic force microscopy studies of living samples, ranging from cells to fresh tissue

Magnetic AC mode (MAC mode) atomic force microscopy (AFM), a novel type of tapping mode AFM in which the cantilever is driven directly by a magnetic field, is a powerful tool for imaging with high spatial resolution and better signal-to-noise in liquid environment. It may largely extend the application of AFM to living samples, especially those are sensitive to cantilever forces, even to multilayer tissue samples. However, there are few reports on the imaging of living cells by MAC mode AFM previously. In our present study, we explore the optimal imaging conditions of MAC mode AFM on living astrocytes and fresh arterial intima surface. We also used nude tips for PicoTREC panel (i.e., Aux in BNC, a new data collecting channel) to image living samples and discussed its difference with phase imaging. We show that living biological samples can be imaged by MAC mode AFM at details of comparable resolution as those by high resolution scanning electron microscopy. Furthermore, the combination of height, amplitude, phase and TREC panel signals provide abundant informations for the characteristics of living samples, such as topography, profile, stiffness and adhesion.     

Topography imaging with a heated atomic force microscope cantilever in tapping mode

This article describes tapping mode atomic force microscopy (AFM) using a heated AFM cantilever. The electrical and thermal responses of the cantilever were investigated while the cantilever oscillated in free space or was in intermittent contact with a surface. The cantilever oscillates at its mechanical resonant frequency, 70.36 kHz, which is much faster than its thermal time constant of 300 micros, and so the cantilever operates in thermal steady state. The thermal impedance between the cantilever heater and the sample was measured through the cantilever temperature signal. Topographical imaging was performed on silicon calibration gratings of height 20 and 100 nm. The obtained topography sensitivity is as high as 200 microVnm and the resolution is as good as 0.5 nmHz(1/2), depending on the cantilever power. The cantilever heating power ranges 0-7 mW, which corresponds to a temperature range of 25-700 degrees C. The imaging was performed entirely using the cantilever thermal signal and no laser or other optics was required. As in conventional AFM, the tapping mode operation demonstrated here can suppress imaging artifacts and enable imaging of soft samples.     

多模态动态原子力显微镜系统

动态原子力显微镜(atomic force microscope,AFM)是通过检测悬臂谐振状态的变化来对物体表面形貌进行测量的。通过对谐振状态的三种因素即振幅、相位、频率的检测,动态AFM可以分为三种工作模式,即振幅反馈、相位反馈与频率反馈模式,这三种反馈模式有着不同的扫描特点。基于硅悬臂具有高阶谐振的特性,动态原子力显微镜可以在悬臂工作于高阶谐振状态时对物体进行扫描。综合上述工作模式研制了一套多模态动态AFM,可以在三种反馈模式、不同阶谐振状态下对物体进行扫描测量。利用该系统在不同反馈模式、不同阶谐振状态下进行了扫描测试,结果显示,系统在各模式下具有亚纳米分辨力,其中在相位反馈模式,悬臂二阶谐振时可达到最优灵敏度与分辨力,分别为17.5V/μm和0.29nm,在最优灵敏度与分辨力状态下对光栅试样进行了三维扫描,得到光栅的三维形貌图。     

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。     

MRT Letter: An extended scanning probe microscopy system for macroscopic topography imaging

Enlightened by the principle of scanning probe microscopy or atomic force microscope (AFM), we proposed a novel surface topography imaging system based on the scanning of a piezoelectric unimorph cantilever. The height of sample surface can be obtained by recording the cantilever's strain using an ultra‐sensitive strain gauge and the Z‐axis movement is realized by electric bending of the cantilever. This system can be operated in the way similar to the contact mode in AFM, with the practical height detection resolution better than 100 nm. Imaging of the inner surface of a steel tube and on a transparent wing of a honey bee were conducted and the obtained results showed that this proposed system is a very promising solution for in situ topography mapping. Microsc. Res. Tech. 77:749–753, 2014. © 2014 Wiley Periodicals, Inc.     

An integrated methodology for data processing in dynamic force spectroscopy of ligand-receptor binding

Dynamic force spectroscopy (DFS), using atomic force microscopy (AFM), is a powerful tool to study ligand-receptor binding. The interaction mode of two binding partners is investigated by exploring stochastic behaviors of bond rupture events. However, to define a rupture event from force-distance measurements is not conclusive or unique in literature. To reveal the influence of event identification methods, we have developed an efficient protocol to manage tremendous amount of data by implementing different choices of peak selection from the force-distance curve. This data processing software simplifies routinely experimental procedures such as cantilever spring constant and force-distance curve calibrations, statistical treatments of data, and analysis distributions of rupture events. In the present work, we took available experimental data from a complex between a chelate metal compound and a monoclonal antibody as a study system.     

Improving tapping mode atomic force microscopy with piezoelectric cantilevers

This article summarizes improvements to the speed, simplicity and versatility of tapping mode atomic force microscopy (AFM). Improvements are enabled by a piezoelectric microcantilever with a sharp silicon tip and a thin, low-stress zinc oxide (ZnO) film to both actuate and sense deflection. First, we demonstrate self-sensing tapping mode without laser detection. Similar previous work has been limited by unoptimized probe tips, cantilever thicknesses, and stress in the piezoelectric films. Tests indicate self-sensing amplitude resolution is as good or better than optical detection, with double the sensitivity, using the same type of cantilever. Second, we demonstrate self-oscillating tapping mode AFM. The cantilever's integrated piezoelectric film serves as the frequency-determining component of an oscillator circuit. The circuit oscillates the cantilever near its resonant frequency by applying positive feedback to the film. We present images and force-distance curves using both self-sensing and self-oscillating techniques. Finally, high-speed tapping mode imaging in liquid, where electric components of the cantilever require insulation, is demonstrated. Three cantilever coating schemes are tested. The insulated microactuator is used to simultaneously vibrate and actuate the cantilever over topographical features. Preliminary images in water and saline are presented, including one taken at 75.5 μm/s—a threefold improvement in bandwidth versus conventional piezotube actuators.     

Influence of antifriction fillers on the surface properties of elastomers based on propylenoxide rubbers

Novel compositions of frost-resistant rubbers (elastomers) are investigated. The possibilities of modifying the basic elastomer using several different fillers are studied. A PTFE-based filler (Forum), technical carbon (TC), and graphene oxide (GO) are tested as fillers. Mechanical, tribological, and microscopic investigations (SEM with elemental analysis and an atomic force microscope (AFM) using different modes) are performed. In order to investigate the operating parameters of these samples, the sample surface was studied before and after the friction tests. The surface topography and the distribution of individual elements over the surface are estimated. Microscopic sections of the samples are studied to obtain information concerning the bulk distribution of the chemical elements. It is shown that the use of fillers such as Forum and technical carbon leads to a better result when they are used together. Their elastic, viscous, and adhesive properties are studied using AFM (the force curve mode—“force-distance curve” and the force modulation mode—“magnitude-distance curve”). It is shown that a distinctive feature of rubber samples having the best tribological properties consists of an increase in rigidity and a decrease in adhesion in the course of tribological testing.     

Explanation and correction of false step heights in amplitude modulation atomic force microscopy measurements on alkane films

We use a prototypical alkane film (n-C(32)H(66) or C32) adsorbed on a SiO(2) surface to compare step heights measured by amplitude modulation atomic force microscopy (AM-AFM) with those measured in the contact mode. The C32 film exhibits layers in which the molecules are oriented with their long axis parallel to the SiO(2) surface followed by partial layers of perpendicular molecules. We show that step heights measured in the AM and contact modes agree in all cases except where the step is between a surface formed by a layer of parallel molecules and one of perpendicular molecules. In this case, the AM mode gives a false step height that is as much as 20% lower than that measured in the contact mode and inferred from synchrotron X-ray specular reflectivity measurements. We propose that the weaker van der Waals forces between the AFM tip and a perpendicular layer compared to a parallel layer causes this discrepancy. We show how to correct the false step height by using the approximately linear relationship observed between phase angle (cantilever oscillation relative to the drive signal) and cantilever height measured in an approach curve.     

Characteristics of a dynamic atomic force microscopy based on a higher-order resonant silicon cantilever and experiments

In order to improve the sensitivity and scanning speed of the dynamic AFM, a surface scanning method using higher-order resonant cantilever is adopted and investigated based on the higher-order resonance characteristics of the silicon cantilever, and the theoretical analysis and experimental verification on the higher-order resonance characteristics of the corresponding dynamic AFM cantilever are given. In this method, the cantilever is excited to oscillate near to its higher-order resonant frequency which is several times higher than that of the fundamental mode. Then the characteristic changes a lot compared with the first-order resonant cantilever. Because of the changes of the quality factor, amplitude and the mode shape of the cantilever, the higher-order resonant AFM gets higher sensitivity and scanning speed. Based on the home-built tapping-mode AFM experiment system, the resolution and the response time of the first and second order resonance measured by experiment are respectively: 0.83 nm, 0.42 nm; 1265 μs, 573 μs. The higher-order resonance cantilever has higher sensitivity and the dynamic measurement performance of the cantilever is significantly improved from the experimental results. This can be a useful method to develop AFM with high speed and high sensitivity. Besides above, the surface profile of a grating sample and its three-dimensional topography are obtained by the higher-order resonant mode AFM.     

Novel operation mode for eliminating influence of inclination angle and friction in atomic force microscopy

The accuracy of topography imaging in contact force mode of atomic force microscopy (AFM) depends on the one-to-one corresponding relationship between the cantilever deflection and the tip–sample distance, whereas such a relationship cannot be always achieved in the presence of friction and incline angle of sample surface. Recently, we have developed a novel operation mode in which we keep the van der Waals force as constant instead of the applied normal force, to eliminate the effect of inclination angle and friction on topography imaging in the contact force mode. We have improved our AFM to enable the new operation mode for validation. Comparative experiments have been performed and the results have shown that the effect of friction and inclination angle on topography imaging in contact mode of AFM can be eliminated or at least decreased effectively by working in the new operation mode we present.     

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.     

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.     

Resonant control of an atomic force microscope micro-cantilever for active Q control

Active Q control may be used to modify the effective quality (Q) factor of an atomic force microscope (AFM) micro-cantilever when operating in tapping mode. The control system uses velocity feedback to obtain an effective cantilever Q factor to achieve optimal scan speed and image resolution for the imaging environment and sample type. Time delay of the cantilever displacement signal is the most common method of cantilever velocity estimation. Spill-over effects from unmodeled dynamics may degrade the closed loop system performance, possibly resulting in system instability, when time delay velocity estimation is used. A resonant controller is proposed in this work as an alternate method of velocity estimation. This new controller has guaranteed closed loop stability, is easy to tune, and may be fitted into existing commercial AFMs with minimal modification. Images of a calibration grating are obtained using this controller to demonstrate its effectiveness.     

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.     

High speed nano-metrology

For manufacturing at the nanometre scale a method for rapid and accurate measurement of the resultant functional devices is required. Although atomic force microscopy (AFM) has the requisite spatial resolution, it is severely limited in scan speed, the resolution and repeatability of vertical and lateral measurements being degraded when speed is increased. Here we present a new approach to AFM that makes a direct and feedback-independent measurement of surface height using a laser interferometer focused onto the back of the AFM tip. Combining this direct height measurement with a passive, feedback-free method for maintaining tip-sample contact removes the constraint on scan speed that comes from the bandwidth of the z-feedback loop. Conventional laser reflection detection is used for feedback control, which now plays the role of minimising tip-sample forces, rather than producing the sample topography. Using the system in conjunction with a rapid scanner, true height images are obtained with areas up to (36 × 36) μm(2) at 1 image/second, suitable for in-line applications.     

Constant tip-surface distance with atomic force microscopy via quality factor feedback

The atomic force microscope (AFM) is a powerful and widely used instrument to image topography and measure forces at the micrometer and nanometer length scale. Because of the high degree of operating accuracy required of the instrument, small thermal and mechanical drifts of the cantilever and piezoactuator systems hamper measurements as the AFM tip drifts spatially relative to the sample surface. To compensate for the drift, we control the tip-surface distance by monitoring the cantilever quality factor (Q) in a closed loop. Brownian thermal fluctuations provide sufficient actuation to accurately determine cantilever Q by fitting the thermal noise spectrum to a Lorentzian function. We show that the cantilever damping is sufficiently affected by the tip-surface distance so that the tip position of soft cantilevers can be maintained within 40 nm of a setpoint in air and within 3 nm in water with 95% reliability. Utilizing this method to hover the tip above a sample surface, we have the capability to study sensitive interactions at the nanometer length scale over long periods of time.     

原子力显微镜在生物分子力学性质方面的研究

原子力显微镜在生物纳米研究领域有广泛应用,包括对生物样品的形貌成像、超微结构、机械性能和相互作用等方面的研究。利用其非修饰和修饰探针进行样品扫描,可以得到样品表面形貌和样品表面某一特定点的力与距离的关系曲线,从而得到相关生物分子的力学性质。目前国际上应用原子力显微镜对生物分子力学特性方面的研究已经成为最热门的研究课题之一,在生物医学和临床医学方面有重要研究意义。本文简述了原子力显微镜的力曲线原理,并对近年来应用原子力显微镜在探测生物分子力学性质方面的研究进展进行了综述。     

Surface modification of AFM silicon probes for adhesion and wear reduction

Tip wear of silicon probes used for an atomic force microscope (AFM) is a critical issue. Wear can result in an increase of tip radius and adhesion between tip and sample, thus reducing the image resolution and introducing artifacts. In order to reduce adhesion, friction, and wear so as to reduce tip related artifacts, liquid lubricant (Z-TETRAOL), self-assembled monolayers (pentafluorophenyltriethoxysilane (PFPTES)), and fluorocarbon polymer (Fluorinert™) were applied on the silicon probe. A comprehensive investigation of adhesion, friction, and wear of the uncoated/coated tips in both ambient air and various humidity levels as well as the influence of the coatings on the image resolution was performed. Experiments showed that the coatings reduced the adhesion, friction, and wear of the silicon tip, improved the initial image resolution, and exhibited less deterioration as compared to that of uncoated tip in the long-term test.