A small scanning tunnelling microscope with large scan range for biological studies

We have developed a scanning tunnelling microscope specially designed for biological applications presenting some new features: the scanner tube is mounted parallel to the surface of the sample which enables a high resolution optical microscope to be brought close to the sample when working in air or liquids. The maximum scan range is 5×20 μm with a vertical range of 20 μm and the total size of the system does not exceed 10×40 mm. The piezo-sensitivity of the scanner tube versus applied voltage was analysed by interferometry measurements and by using scanning tunnelling microscopes. We found a value for the piezoelectric constant d₁₃ of ?1·71 Å/V at low voltages (under a few volts) going up to ?2 Å/V for higher voltages. Large-scale images of a carbon grid showed a surprisingly good linearity of the scanner tube.     

Arrangement and stability of atoms at the apex of a scanning tip

A field ion microscope was used to examine the stability of the atomic arrangement at tip apexes. Although a single W atom at the top layer of a [111]-orientated tip apex protrudes from the underneath layer by only 0·91 Å, the present study suggests that the (111)-orientated W tip is the most desirable tip for scanning tunnelling microscopes because a large activation energy for surface diffusion on the (111) plane immobilizes the apex atom while the tip scans over a specimen surface and the tip apex can be resharpened by simply heating the tip.     

Investigation of intrinsic crystalline Si(111) and amorphous silicon surfaces in air with STM

Crystalline (111) and amorphous silicon surfaces have been studied by scanning tunnelling microscopy. An orientated, reproducible, corrugated structure has been observed on Si(111) surfaces. The voltage dependence of the corrugation amplitude may be attributable to surface states. The surfaces of amorphous silicon thin films show some reproducible structure in the range of a few tens of ångströms, observable only when the applied voltage between the tip and sample is between ?1·3 and +0·4V.     

Electrical detection of β-amyloid (1-40) using scanning tunneling microscopy

Numerous studies have shown that the presence of β-amyloid (1-40) in cerebrospinal fluid can be used as a potential biomarker for Alzheimer's disease. Identifying biomarkers for Alzheimer's disease is highly important because these biomarkers could be used to establish the diagnosis before the disease reaches clinical severity. In this study, a vertically configured electrical detection system associated with scanning tunneling microscopy (STM) was used to characterize antigen–antibody binding interactions. The proposed technique can be easily utilized to construct a multiple measurement system in a protein chip. The immunocomplexes used in the model protein comprise β-amyloid (1-40), corresponding antibody fragments, and gold nanoparticle–antibody conjugates. The electrical tunneling current between the STM tip and these complexes exhibited a peak-like pulse, where the frequency of these pulses was dependent on the surface density of bound complexes. Hence, a quantitative measurement of β-amyloid concentration from a periodogram analysis of peak frequency was successfully achieved at concentrations as low as 1 fg/mL.     

一种基于剪切力的距离控制方法在扫描近场光学显微镜中的应用

介绍了扫描近场光学显微镜中基于剪切力的样品、探针间距离控制的方法。当受振动激励的光纤探针由远处逐渐接近样品表面时,样品与针尖间的剪切力使针尖的振动振幅减小,通过检测探针振幅的变化从而控制针尖与样品间的距离。此种方法可以方便地将光纤探针导入工作区域内并在扫描过程中保持适当的高度。我们测量了探针系统的幅频特性和力曲线,并用该方法获得4μm×4μm的范围内光盘表面的形貌信息。     

Calibration of the scanning (atomic) force microscope with gold particles

Scanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.     

Application of atomic force microscopy in bacterial research

The atomic force microscope (AFM) has evolved from an imaging device into a multifunctional and powerful toolkit for probing the nanostructures and surface components on the exterior of bacterial cells. Currently, the area of application spans a broad range of interesting fields from materials sciences, in which AFM has been used to deposit patterns of thiol‐functionalized molecules onto gold substrates, to biological sciences, in which AFM has been employed to study the undesirable bacterial adhesion to implants and catheters or the essential bacterial adhesion to contaminated soil or aquifers. The unique attribute of AFM is the ability to image bacterial surface features, to measure interaction forces of functionalized probes with these features, and to manipulate these features, for example, by measuring elongation forces under physiological conditions and at high lateral resolution (<1 Å). The first imaging studies showed the morphology of various biomolecules followed by rapid progress in visualizing whole bacterial cells. The AFM technique gradually developed into a lab‐on‐a‐tip allowing more quantitative analysis of bacterial samples in aqueous liquids and non‐contact modes. Recently, force spectroscopy modes, such as chemical force microscopy, single‐cell force spectroscopy, and single‐molecule force spectroscopy, have been used to map the spatial arrangement of chemical groups and electrical charges on bacterial surfaces, to measure cell–cell interactions, and to stretch biomolecules. In this review, we present the fascinating options offered by the rapid advances in AFM with emphasizes on bacterial research and provide a background for the exciting research articles to follow. SCANNING 32: 74–96, 2010. © 2010 Wiley Periodicals, Inc.     

Adsorbate deformation as a contrast mechanism in STM images of biopolymers in an aqueous environment: images of the unstained,hydrated DNA double helix

A stable residual aggregate remains on a submerged gold surface after electrophoretic deposition of DNA. We present scanning tunnelling microscope (STM) images of these aggregates which show many objects with the geometry of DNA, clearly displaying the 3·4 nm helix pitch. These images are quite distinctive, and cannot be generated when the deposition technique is used without DNA in the buffer solution. A characteristic of these images is that the tip is observed to dip down over the DNA molecule at the same time as the apparent barrier height drops by a factor of about four. The tip displacement is accounted for by a model in which contrast is dominated by local fluctuations in the deformability of the adsorbate layer, a quantity deduced from measurements of the apparent barrier heights in air, water, over small molecule adsorbates, and over DNA.     

Atomic force microscope imaging and force measurements at electrified and actively corroding interfaces: challenges and novel cell design

We report the design of an improved electrochemical cell for atomic force microscope measurements in corrosive electrochemical environments. Our design improvements are guided by experimental requirements for studying corrosive reactions such as selective dissolution, dealloying, pitting corrosion, and∕or surface and interface forces at electrified interfaces. Our aim is to examine some of the limitations of typical electrochemical scanning probe microscopy (SPM) experiments and in particular to outline precautions and cell-design elements, which must necessarily be taken into account in order to obtain reliable experimental results. In particular, we discuss electrochemical requirements for typical electrochemical SPM experiments and introduce novel design features to avoid common issues such as crevice formations; we discuss the choice of electrodes and contaminations from ions of reference electrodes. We optimize the cell geometry and introduce standard samples for electrochemical AFM experiments. We have tested the novel design by performing force-distance spectroscopy as a function of the applied electrochemical potential between a bare gold electrode surface and a SAM-coated AFM tip. Topography imaging was tested by studying the well-known dealloying process of a Cu(3)Au(111) surface up to the critical potential. Our design improvements should be equally applicable to in situ electrochemical scanning tunneling microscope cells.     

Feedback-controlled laser fabrication of micromirror substrates

Short (40-200 μs) single focused CO(2) laser pulses of energy ?100 μJ were used to fabricate high quality concave micromirror templates on silica and fluoride glass. The ablated features have diameters of ≈20-100 μm and average root-mean-square (RMS) surface microroughness near their center of less than 0.2 nm. Temporally monitoring the fabrication process revealed that it proceeds on a time scale shorter than the laser pulse duration. We implement a fast feedback control loop (≈20 kHz bandwidth) based on the light emitted by the sample that ensures an RMS size dispersion of less than 5% in arrays on chips or in individually fabricated features on an optical fiber tip, a significant improvement over previous approaches using longer pulses and open loop operation.     

Surface potential study of Au(111) surfaces

The structure of the one-dimensional surface potential barrier of the gold (111) surface has been investigated by the analysis of the deflection of the central spot of a microdiffraction pattern which is easily obtained by using a HB-5 scanning transmission electron microscope. The result shows direct evidence for the existence of this surface barrier potential up to about 25 Å away from surface. From a suggested modified image potential barrier model, the streaking length on the central spot has been obtained from calculation and compared with the experimental measurement. The agreement is good within the limitations of the experimental technique.     

The future of the electron microscope*

The future of electron microscopy lies as much with the conventional as with the newer instruments. The point resolving power of the latter is likely to be pushed to 1 Å, but only after a considerable effort in solving problems of mechanical and electrical stability. Progress in correcting the lens aberrations is even slower. Techniques of specimen preparation and microscope operation are continuously being improved, but will need even greater refinement if proper use is to be made of a 1 Å resolving power, e.g. for identifying bases in a nucleic acid molecule. Extension of the working voltage to 1 MeV and above is increasing the usefulness of the conventional electron microscope, particularly in metallurgy, and plans are now being made for even higher voltages. Of the newer, unconventional instruments, the scanning electron microscope is already establishing a place for itself, especially in industrial applications where surface conditions on the microscale are important. It is likely to find increasing use in micro-circuitry, but also in some branches of biological research, even if its resolving power cannot be brought below 100 Å. The combination of X-ray spectrometry with electron microscopy holds promise of wide application, in the form of a hybrid electron microscope-microanalyser. Secondary-emission microscopes are slowly finding a place for themselves, mainly in applied science and technology. Mirror microscopes, for surface investigations, and ion microscopes, for microanalysis, are still in an early stage but have interesting possibilities. The field ion microscope, simplest in principle but sophisticated in technique, is unique in showing the position of individual atoms in a metal tip. It is already finding applications, especially in studies of radiation damage.     

Pulsed field evaporation of ions from polar solutions

An implementation of a technique for studying the release of ions from polar solutions under the action of electric field pulses is presented. A track membrane with nanoscale channels is used in this technique to stabilize the solution surface. The dependences of the ion yield on the parameters of extracting voltage pulses, as well as on the KI salt concentration in water?glycerol solutions have been obtained. The main advantage of the use of short pulses over a constant voltage technique is the possibility of expanding the range of the field strengths extracting ions from a solution without the risk that the solution leaks to the vacuum side of the membrane. In addition, a high stability of the extraction process is provided and the continuous operating time without membrane replacement is increased. The formation of the extraction field at the end of the channel due to the tip effect provides a fast start of the extraction process immediately after applying a voltage pulse, which also simplifies work with the membrane interface.     

Morphological Growth of Sputtered MoS2 Films

Sputtered MoS₂ films from 300 Å to 20,000 Å thick were deposited on metal and glass surfaces. The substrate effects such as surface temperature, finish, pretreatment, and chemistry as they affect the film formation characteristics were investigated by optical, electron transmission, electron diffraction, and scanning electron microscopy. Substrate temperature and surface chemistry were found to be the prime variables as to the formation of a crystalline or amorphous film. The friction characteristics are strictly influenced by the type of film formed. Surface chemistry and surface pretreatment account for compound formation and corresponding grain growth, which directly affect the adhesion characteristics, resulting in poor adherence. The type of surface finish (topography) as related to scratches, impurities, inhomogeneities, etc., are favorable nucleation sites for the growth of isolated and complex nodules within the film, and various complex surface overgrowths on the film. These nodular growth features have progressively more undesirable effects on the film behavior as the film thickness increases.     

扫描隧道显微镜系统的优化研究

对扫描隧道显微镜(STM)系统进行了优化研究,实现了对环境振动的隔绝和电噪声的屏蔽,并在此基础上,设计并完成CCD显微监测系统,实现了对样品—探针逼近过程的实时临控。对整个系统进行了联合调试,成功获得高定向热解石墨和金表面的结构图像。     

Apertureless near-field Raman spectroscopy

We report on the tip‐enhanced Raman spectra of C60 obtained on a custom‐built apertureless scanning near‐field optical microscope. A commercial atomic force microscope tip coated with 100 nm thickness of gold was used to enhance locally the Raman signal and permit topographic and spectral information to be acquired simultaneously. We present preliminary data which demonstrate the tip enhancement effect using C60 as a test sample.     

Guiding self-assembly with the tip of an atomic force microscope

We report the guided self-assembly of nanoparticles to geometrically well-defined charge patterns written on a dielectric surface with the conductive tip of an atomic force microscope (AFM). Charges are deposited in 30-90-nm thick fluorocarbon layers by applying voltage pulses to the conductive AFM tip. The samples are being developed by dipping them into an organic suspension of silica nanoparticles. Coulomb forces draw the nanoparticles to the charge patterns. With this simple process, we achieve a resolution of about 800 nm.     

Electrically conductive and optically transparent Sb-doped SnO2 STM-probe for local excitation of electroluminescence

We demonstrate that an optically transparent and electrically conductive antimon-doped tin-oxide tip that is prepared in a sol-gel process can be used as a probe for scanning tunnelling microscopy (STM), yielding atomic vertical and nanometre lateral resolution. Emission of visible light from the tunnelling junction between gold particles and the tip is observed for bias voltages above 7 V. In contrast to the metallic tips generally used in STM, this tip does not significantly perturb the local optical response. Therefore, the tunnelling induced light can be used to map the optical near-field of surface structures with the tunnel gap acting as highly localised light source for the investigation of near-field enhancement in complex metal structures.     

Nano-machining of gold and semiconductor surfaces

Using a scanning tunnelling microscope tip formed by cutting a platinum wire, we have modified the surfaces of gold and Hg_1-xCd_xTe on a nanometre scale by mechanical contact between the tip and the surface. By using the same tip to form images, we have been able to gain ‘before’ and ‘after’ pictures of surfaces that have been selectively ‘sanded’, controllably ‘chiselled’, and ‘swept’. We have also obtained images taken under glycerin of Hg_1-xCd_xTe and of the diluted magnetic semiconductor Hg_1-xMn_xTe.     

STM imaging of noble metal electrodes in solution under potentiostatic control

We have combined a three-electrode cell arrangement with a scanning tunnelling microscope (STM) to image the surfaces of polycrystalline noble metal electrodes under potentiostatic conditions. STM imaging was made in the potential range where a tip and the electrodes are ideally polarized. We have observed on a submicron scale that the electrochemically roughened Ag electrode surface relaxed over times of 40 min at ?0·16 V versus SCE in 0·1 m KCl. We have also imaged a dynamic process (formation and dissociation) of the submicron-scale reconstruction of the Au electrode subjected to electrochemical oxidation-reduction cycles. The results can be explained by the absorption of Cl^? ions and the surface diffusion of adatoms or clusters. STM imaging under potentiostatic control leads to the discovery of phenomena occurring at solid metal/electrolyte interfaces.