Assembling and imaging of his-tag green fluorescent protein on mica surfaces studied by atomic force microscopy and fluorescence microscopy

The adsorption of his-tag green fluorescent protein (GFPH(6)) onto the mica surfaces has been studied by atomic force microscopy (AFM) and laser confocal fluorescence microscopy. By controlling the adsorption conditions, separated single GFPH(6) and GFPH(6) monolayer can be adsorbed and formed on mica surfaces. In present experiments, based on the AFM measurement, we found that the adsorbed GFPH(6) was bound on the mica surface with its beta-sheets. The formed GFPH(6) monolayer on mica surfaces was flat, uniform, and stable. Some applications of the formed monolayer have been demonstrated. The formed monolayer can be used as a substrate for DNA imaging and AFM mechanical lithography.     

Analysis of simulated scanning of atomic-scale silicon surface by atomic force microscopy

This study constructs a contact-mode atomic force microscopy (AFM) simulation measurement model with constant force mode to simulate and analyze the outline scanning measurement by AFM. The simulation method is that when the probe passes the surface of sample, the action force of the atom of sample received by the atom of the probe can be calculated by using Morse potential. Through calculation, the equivalent force on the cantilever of probe can be acquired. By using the deflection angle equation for the cantilever of probe developed and inferred by this study, the deflection angle of receiving action force can be calculated. On the measurement point, as the deflection angle reaches a fixed deflection angle, the scan height of this simulation model can be acquired. By scanning in the right order, the scan curve of the simulation model can be obtained. By using this simulation measurement model, this study simulates and analyzes the scanning of atomic-scale surface outline. Meanwhile, focusing on the tip radii of different probes, the concept of sensitivity analysis is employed to investigate the effects of the tip radius of probe on the atomic-scale surface outline. As a result, it is found from the simulation on the atomic-scale surface that within the simulation scope of this study, when the tip radius of probe is greater than 12 nm, the effects of single atom on the scan curve of AFM can be better decreased or eliminated.     

Nanostructures in silicon investigated by atomic force microscopy and surface photovoltage spectroscopy

Surface photovoltage spectroscopy (SPS) and conductive atomic force microscopy (C-AFM) have been used for the characterization of nanocrystalline hydrogenated Si (nc-Si:H). This is a promising material both for silicon-based opto-electronics as well as for photovoltaic applications. Notwithstanding its interesting properties many issues regarding the material electronic and optical properties are not completely understood. The present contribution reports microscopic and spectroscopic analyses of nc-Si:H films grown for photovoltaic applications by low-energy plasma-enhanced chemical vapor deposition technique. Electronic levels associated with defect states were investigated by SPS, whereas the conduction mechanism at a microscopic level was investigated by C-AFM.     

Towards quantification of doping in gallium arsenide nanostructures by low-energy scanning electron microscopy and conductive atomic force microscopy

We calculate a universal shift in work function of 59.4 meV per decade of dopant concentration change that applies to all doped semiconductors and from this use Monte Carlo simulations to simulate the resulting change in secondary electron yield for doped GaAs. We then compare experimental images of doped GaAs layers from scanning electron microscopy and conductive atomic force microscopy. Kelvin probe force microscopy allows to directly measure and map local work function changes, but values measured are often smaller, typically only around half, of what theory predicts for perfectly clean surfaces.     

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.     

Evaluation of surface roughness and nanostructure of indium tin oxide (ITO) films by atomic force microscopy

Indium tin oxide was deposited on a glass (soda lime glass) by radiofrequency sputtering system at different sputtering gas (argon/oxygen 90/10%) pressures (20-34 mTorr) at room temperature. The sputtering rate was affected by the sputtering gas pressure. The optimum sputtering gas pressure was found to be 27 mTorr. The samples at different thicknesses (168, 300, 400, 425, 475, 500 and 630 nm) were deposited on the substrate. Transparency, electrical conductivity and surface roughness of the films were characterized. The samples were annealed at 350, 400 and 450 degrees C to evaluate annealing process effects on the concerned parameters and, therefore, the above-mentioned measurements were repeated again. The films exhibited reasonable optical transmittance and electrical conductivity and greatly improved after annealing. The characterization was focused on the scanning of the film surfaces before and after annealing, which has a prominent effect on the optical properties of the films. Film surfaces were scanned by scanning probe microscopy in contact atomic force mode. The most consideration was devoted to image analysis.     

Imaging and studying human topoisomerase I on mica surfaces in air and in liquid by atomic force microscopy

In this study, the topography of human topoisomerase I (TOPO I) on mica surfaces in air and in liquid has been studied by atomic force microscopy (AFM). The average height of TOPO I on mica surface in air measured by AFM was 2.59±0.32 nm. After adsorption of the 0.3 U/µl TOPO I on mica surfaces for 2 h, and then imaged in liquid by AFM, well‐separated single TOPO I was observed. The average height of TOPO I on mica surfaces in liquid measured by AFM was 2.93±0.42 nm. After adsorption of the 4 U/µl TOPO I on mica surfaces for 1.5 h, TOPO I monolayer can be formed. The produced TOPO I monolayer on mica was flat and exhibited good stability. SCANNING 31: 160–166, 2009. © 2009 Wiley Periodicals, Inc.     

Polysaccharide properties probed with atomic force microscopy

In recent years, polysaccharides have been extensively studied using atomic force microscopy (AFM). Owing to its high lateral and vertical resolutions and ability to measure interaction forces in liquids at pico‐ or nano‐Newton level, the AFM is an excellent tool for characterizing biopolymers. The first imaging studies showed the morphology of polysaccharides, but gradually more quantitative image analysis techniques were developed as the AFM grew easier to use in aqueous liquids and in non‐contact modes. Recently, AFM has been used to stretch polysaccharides and characterize their physicochemical properties by application of appropriate polymer stretching models, using a technique called single‐molecule force spectroscopy. From application of such models as the wormlike chain, freely jointed chain, extensible‐freely jointed chain, etc., properties such as the contour length, persistence length and segment elasticity or spring constant can be calculated for polysaccharides. The adhesion between polysaccharides and surfaces has been quantified with AFM, and this application is particularly useful for studying polysaccharides on microbial and other types of cells, because their adhesion is controlled by biopolymer characteristics. This review presents a synthesis of the theory and techniques currently in use to probe the physicochemical properties of polysaccharides with AFM.     

Immobilization and condensation of DNA with 3-aminopropyltriethoxysilane studied by atomic force microscopy

We used different methods to modify a mica surface with 3-aminopropyltriethoxysilane (APTES), and then used it as substrate to immobilize DNA for atomic force microscopy (AFM) observation. The evaporation method and solution modifying method were investigated and evaluated. The solution modifying method was found to be relatively simple and effective. Using an APTES solution-modified mica surface, DNA immobilization appeared more reproducible and it could be imaged in liquid. The mixed solution of APTES and DNA was dropped directly onto the mica surface for AFM imaging. We found that DNA can condense in APTES water solutions. Toroids, rods and intermediate structures of condensation were captured by AFM.     

Dynamic behavior of amplitude detection Kelvin force microscopy in ultrahigh vacuum

The acquisition rate of all scanning probe imaging techniques with feedback control is limited by the dynamic response of the control loops. Performance criteria are the control loop bandwidth and the output signal noise power spectral density. Depending on the acceptable noise level, it may be necessary to reduce the sampling frequency below the bandwidth of the control loop. In this work, the frequency response of a vacuum Kelvin force microscope with amplitude detection (AM-KFM) using a digital signal processing (DSP) controller is characterized and optimized. Then, the main noise source and its impact on the output signal is identified. A discussion follows on how the system design can be optimized with respect to output noise. Furthermore, the interaction between Kelvin and distance control loop is studied, confirming the beneficial effect of KFM on topography artefact reduction in the frequency domain. The experimental procedure described here can be generalized to other systems and allows to locate the performance limitations.     

Investigation of nanoparticles by atomic and lateral force microscopy

Metallic nanoparticles have been produced on vitreous carbon substrates by means of thermal evaporation. From pictures of the particles, made by a high-resolution scanning electron microscope (HRSEM), a semispherical shape is suggested due to the total mass of deposited material. Atomic force microscopy (AFM) has been applied to this sample in order to get direct topographic information. The AFM has been operated with normal and super tips, the latter having a smaller cone angle and radius, thus following more precisely the contours of an object. Simultaneously lateral-force microscopic (LFM) images have been recorded. Major differences between the contents of HRSEM- and AFM-images are considered, emphasizing the important influence of the tips' geometry. Both the AFM and LFM line scans have been compared with and have qualitatively agreed with those calculated under simplifying assumptions.     

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.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

Visualization of cellular DNA crosslinks by atomic force microscopy

Cellular DNA crosslinks are a type of DNA damage induced by toxic chemicals or high‐energy radiation. If damaged DNA is not rapidly repaired, cells will die or mutate. To evaluate the types of DNA damage and their influence on vital cell activities, it is necessary to be able to detect DNA crosslinks. To date, indirect methods such as alkaline elution, potassium chloride–sodium dodecyl sulfate assay and comet assay have been used to detect DNA damage. Direct morphological observation, on the other hand, may be a useful tool to differentiate the types of DNA damage. In this report, atomic force microscopy (AFM) has been employed to visualize the breakage and crosslinking of cellular DNA strands in cells treated with formaldehyde and hydrogen peroxide. Our results showed that toxic chemical‐induced crosslinking of cellular DNA occurred in a dose‐dependent manner. DNA conglomerates were observed with high concentrations of formaldehyde, and the AFM observations were consistent with those of a comet assay. Our experiments demonstrate that AFM is an efficient method to differentiate the types of DNA damage. SCANNING 31: 75–82, 2009. © 2009 Wiley Periodicals, Inc.     

Large-scale, uniform DNA network on 11-mercaptoundecanoic acid modified gold (111) surface: atomic force microscopy study

Large-scale, uniform plasmid deoxyribonucleic acid (DNA) network has been successfully constructed on 11-mercaptoundecanoic acid modified gold (111) surface using a self-assembly technique. The effect of DNA concentration on the characteristics of the DNA network was investigated by atomic force microscopy. It was found that the size of meshes and the height of fibers in the DNA network could be controlled by varying the concentration of DNA with a constant time of assembly of 24 h.     

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.     

The study on the atomic force microscopy base nanoscale electrical discharge machining

This study proposes an innovative atomic force microscopy (AFM) based nanoscale electrical discharge machining (AFM-based nanoEDM) system which combines an AFM with a self-produced metallic probe and a high-voltage generator to create an atmospheric environment AFM-based nanoEDM system and a deionized water (DI water) environment AFM-based nanoEDM system. This study combines wire-cut processing and electrochemical tip sharpening techniques on a 40-μm thick stainless steel sheet to produce a high conductive AFM probes, the production can withstand high voltage and large current. The tip radius of these probes is approximately 40 nm. A probe test was executed on the AFM using probes to obtain nanoscales morphology of Si wafer surface. The silicon wafer was as a specimen to carry out AFM-base nanoEDM process in atmospheric and DI water environments by AFM-based nanoEDM system. After experiments, the results show that the atmospheric and DI water environment AFM-based nanoEDM systems operate smoothly. From experimental results, it can be found that the electric discharge depth of the silicon wafer at atmospheric environments is a mere 14.54 nm. In a DI water environment, the depth of electric discharge of the silicon wafer can reach 25.4 nm. This indicates that the EDM ability of DI water environment AFM-based nanoEDM system is higher than that of atmospheric environment AFM-based nanoEDM system. After multiple nanoEDM process, the tips become blunt. After applying electrochemical tip sharpening techniques, the tip radius can return to approximately 40 nm. Therefore, AFM probes produced in this study can be reused.     

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.     

Atomic vacancy-induced friction on the graphite surface: observation by lateral force microscopy

Lateral force microscopy has been employed to investigate the frictional behaviour of atomic vacancies on the graphite surface. Such a study was only made possible by the controlled expansion of originally single‐atom vacancies into multiatom vacancies, employing oxygen plasma etching for this purpose. Enhanced friction was observed on the vacancy regions compared with pristine areas of graphite, the origin of which is examined and discussed.