A large-sample atomic force microscope observing in both air and liquid

A large-sample atomic force microscope (AFM) that allows high resolution observation in both air and liquid has been developed. With a unique beam tracking method, laser beam is capable of reflecting off the same spot on the AFM cantilever throughout raster scan over the entire scan area, either operating in air or in liquid environment. Incorporating the stand-alone AFM probe unit with an automated large sample stage, wide-scan-range imaging can be realized with high resolution and slight distortion. In addition, an image stitching method is utilized to build a broad merged image with range up to millimeters while keeping nanometer order resolution. By using a large-volume liquid bath, large and massive sample can be observed in liquid with this AFM system. Several typical experiments have been carried out to demonstrate the imaging ability and stability of this AFM. Topographic structures of gold pattern on a glass substrate are scanned at two different places on the same specimen surface. The porosity of a sheet of filter paper is then characterized in both air and water. Finally, larger-area AFM image of anodic aluminum oxide template in oxalic acid is on spot obtained by merging several individually scanned images together. Experiments show that this AFM system can offer high resolution and wide range AFM images even for large samples with remarkable capabilities in various environments.     

气/液两用型原子力显微镜及其应用研究

发展了一种气/液两用型原子力显微镜(AFM)系统,讨论了其工作原理,给出了优化的气/液两用型探头及扫描与反馈控制电路。利用该型AFM系统分别对大气环境下的硅片以及液体环境下的刀片和多孔氧化铝样品进行了扫描检测。实验结果表明,该型AFM系统在大气和液体环境中均可扫描获得理想的AFM图像,分辨力达到纳米量级,扫描范围可达4000nm×4000nm,可满足各种微纳米扫描测量的要求。     

Aspect-ratio and lateral-resolution enhancement in force microscopy by attaching nanoclusters generated by an ion cluster source at the end of a silicon tip

One of the factors that limit the spatial resolution in atomic force microscopy (AFM) is the physical size of the probe. This limitation is particularly severe when the imaged structures are comparable in size to the tip's apex. The resolution in the AFM is usually enhanced by using sharp tips with high aspect ratios. In the present paper we propose an approach to modify AFM tips that consists of depositing nanoclusters on standard silicon tips. We show that the use of those tips leads to atomic force microscopy images of higher aspect ratios and spatial resolution. The present approach has two major properties. It provides higher aspect-ratio images of nanoscale objects and, at the same time, enables to functionalize the AFM tips by depositing nanoparticles with well-controlled chemical composition.     

A high-pressure atomic force microscope for imaging in supercritical carbon dioxide

A high-pressure atomic force microscope (AFM) that enables in situ, atomic scale measurements of topography of solid surfaces in contact with supercritical CO(2) (scCO(2)) fluids has been developed. This apparatus overcomes the pressure limitations of the hydrothermal AFM and is designed to handle pressures up to 100 atm at temperatures up to ～350 K. A standard optically-based cantilever deflection detection system was chosen. When imaging in compressible supercritical fluids such as scCO(2), precise control of pressure and temperature in the fluid cell is the primary technical challenge. Noise levels and imaging resolution depend on minimization of fluid density fluctuations that change the fluid refractive index and hence the laser path. We demonstrate with our apparatus in situ atomic scale imaging of a calcite (CaCO(3)) mineral surface in scCO(2); both single, monatomic steps and dynamic processes occurring on the (1014) surface are presented. This new AFM provides unprecedented in situ access to interfacial phenomena at solid-fluid interfaces under pressure.     

Observation of HOPG by STM and contact AFM in various gas atmospheres under pressures up to 1.1 MPa

Apparatus comprising a scanning tunneling microscopy (STM) and an atomic force microscopy (AFM) has been developed for use under supra-atmospheres. Observations of highly oriented pyrolytic graphite (HOPG) were carried out by STM and contact AFM operating in air and various gas atmospheres (hydrogen, helium, neon and argon) under pressures up to 1.1 MPa. Atomic resolution images of the HOPG were obtained by STM in all the gas atmospheres studied. However, it was found that the presence of water vapor gave rise to a noise current at increased pressures. Using contact AFM, the atomic resolution in an argon atmosphere decreased with increasing pressure, while atomic images were obtained under the other gas atmospheres at 1.1 MPa.     

Finite-element vibration analysis of tapping-mode atomic force microscopy in liquid

Investigation of morphology and mechanical properties of biological specimens using atomic force microscopy (AFM) often requires its operation in liquid environment. Due to the hydrodynamic force, the vibration of AFM cantilevers in liquid shows dramatically different dynamic characteristics from that in air. A good understanding of the dynamics of AFM cantilevers vibrating in liquid is needed for the interpretation of scanning images, selection of AFM operating conditions, and evaluation of sample's mechanical properties. In this study, a finite element (FE) model is used for frequency and transient response analysis of AFM cantilevers in tapping mode (TM) operated in air or liquid. Hydrodynamic force exerted by the fluid on AFM cantilevers is approximated by additional mass and hydrodynamic damping. The additional mass and hydrodynamic damping matrices corresponding to beam elements are derived. With this model, numerical simulations are performed for an AFM cantilever to obtain the frequency and transient responses of the cantilever in air and liquid. The comparison between our simulated results and the experimentally obtained ones shows good agreement. Based on the simulations, different characteristics of cantilever dynamics in air and liquid are discussed.     

Real-time displacement measurements with a Fabry-Perot cavity and a diode laser

We present the basic operating principles of a traceable measurement system suitable for use with atomic force microscopes (AFMs) and nanometer-resolution displacement sensors. Our method is based upon a tunable external-cavity diode laser system which is servo-locked via a phase-modulated heterodyne locking technique to a Fabry-Perot interferometer cavity. We discuss mechanical considerations for the use of this cavity as a displacement metrology system and we describe methods for making real-time (sub 10 ms sampling period) measurements of the optical heterodyne signals. Our interferometer system produces a root-mean-squared (RMS) displacement measurement resolution of 20 pm. Two applications of the system are described. First, the system was used to examine known optical mixing errors in a heterodyne Michelson interferometer. Second, the Fabry-Perot interferometer was integrated into the Z axis of a commercial AFM scanning stage and used to produce interferometer-based images of a 17 nm step height specimen. We also demonstrate atomic resolution interferometer-based images of a 0.3 nm silicon single atomic step-terrace specimen.     

Atomically resolved imaging by low-temperature frequency-modulation atomic force microscopy using a quartz length-extension resonator

Low-temperature ultrahigh vacuum frequency-modulation atomic force microscopy (AFM) was performed using a 1 MHz length-extension type of quartz resonator as a force sensor. Taking advantage of the high stiffness of the resonator, the AFM was operated with an oscillation amplitude smaller than 100 pm, which is favorable for high spatial resolution, without snapping an AFM tip onto a sample surface. Atomically resolved imaging of the adatom structure on the Si(111)-(7x7) surface was successfully obtained.     

补偿压电陶瓷迟滞和蠕变的逆控制算法

研究了用于扫描探针显微镜,特别是原子力显微镜(AFM)中的扫描器高精度定位问题。压电陶瓷驱动器通常用于这种扫描器中,在无补偿的开环控制期间,它在输入电压和输出位移之间表现出明显的迟滞和蠕变。迟滞和蠕变降低了扫描器的定位精度并使扫描图像出现了畸变。给出了迟滞和蠕变模型及参数的在线辨识方法。在AFM的扫描控制中,使用基于该模型的逆控制算法补偿了压电陶瓷的迟滞和蠕变。在分析中,Preisach迟滞模型和对数蠕变模型被用于描述压电陶瓷驱动器的非线性。由于该方法不需要在控制过程中进行参数设置,因此很容易使用。此外由于是开环控制方法,可以具有更好的分辨率。闭环操作能够给出较好的迟滞和蠕变补偿,但由于在高带宽情况下传感器动态范围的限制,对于小扫描区域或样本特征,它减小了成像的分辨率。三角波轨迹跟踪的仿真结果说明轨迹误差在传感器噪声量级上,证明了这个方法的有效性。     

High resolution imaging of surface patterns of single bacterial cells

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.     

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion.

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.     

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside‐out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X‐100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high‐resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high‐resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.     

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.     

Dynamic force microscopy imaging of native membranes

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.     

Combined atomic force and scanning reflection interference contrast microscopy

A sphere attached to a cantilever is used simultaneously as an atomic force microscope (AFM) tip and as a curved reflective surface for producing scanning reflection interference contrast microscope (RICM) images of fluorescent beads dried onto a glass slide. The AFM and RICM images are acquired in direct registration which enables the identification of individually excited beads in the AFM images. The addition of a sharp, electron beam-deposited tip to the sphere gives nanometer resolution AFM images without loss of optical contrast.     

Recent advances in atomic force microscopy of DNA

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200–500 picograms per sample. Second, the new “tapping” mode has been used to improve the ease and resolution of AFM-imaging of DNA in air. Finally, AFM images are presented of single-stranded ΦX-174 virion DNA with the gene 32 single-stranded binding protein. A summary of the current state of the field and of the methods for preparing and imaging DNA in the AFM is also presented.     

Voltage preamplifier for extensional quartz sensors used in scanning force microscopy

Extensional-mode quartz resonators are being increasingly used as force sensors in dynamic scanning force microscopy or atomic force microscopy (AFM). We propose a voltage preamplifier in order to amplify the charge induced on quartz electrodes. The proposed solution has some advantages over the typically used current-to-voltage converters. First, the gain does not depend on the inner parameters of the quartz resonator, which are usually unknown for the specific resonator and may even vary during the measurement. Second, with such an amplifier a better signal-to-noise ratio can be achieved. Finally, we present AFM images of the Si(111) and the SiO(2) surfaces obtained by the voltage preamplifier with simultaneously recorded tunneling current.     

Use of AFM for imaging and measurement of the mechanical properties of light-convertible organelles in plants

We obtained topographic images of etioplasts and chloroplasts and measured their elasticity in a physiological buffer using an atomic force microscope (AFM) and found a possible correlation between the morphological and mechanical properties during the light conversion of etioplasts to chloroplasts. Alcian blue 8GX dye was found to be effective for immobilizing the plant organelles stably on a glass surface for AFM experiments. We employed the tapping-mode AFM with a cantilever soft enough to measure the elasticity of the organelles in a liquid solution. The best images of soft, spherical organelles were obtained using the tapping-mode AFM with oscillation at the thermal vibration frequency of the cantilever of around 3 kHz. Whereas etioplasts were found to be smooth-surfaced and stiff against compression by the AFM tip, before light conversion to chloroplasts, they became rough-surfaced and mechanically soft after exposure to light. The elasticity of etioplasts was 20 times higher than that of chloroplasts, probably reflecting changes in their inner structures.     

Accurate measurement of the out-of-plane motion of a tip-scanning atomic force microscope

A tip-scanning atomic force microscope (AFM) can be used as a highly accurate height-measuring instrument for large samples, such as liquid crystal displays. To accurately measure the flatness or surface roughness of large samples, the xy-scanner-induced out-of-plane motion must be known to discriminate scanner artifacts from the measured AFM images. As the topographic signals of AFM measurements contain the hysteresis of the z-scanner piezoelectric actuators, actual movements of the z-scanner were measured using a z-axis sensor glued to the actuator. The actual out-of-plane motion of the xy-scanner was found to be less than 1 nm for a 50-μm scan.