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.     

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.     

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.     

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica

Multivalent ions in solution are known to mediate attraction between two like‐charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either ‘salt bridging’ or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re‐explore current theories of like‐charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.     

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.     

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.     

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.     

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.     

Disassembly of DNA–ligand on mica surface: atomic force microscopy studies

Disassembly of DNA–ligands, including DNA–methylene blue (MB) complex and DNA–Co(phen)₃³⁺ complex on mica surface, were investigated by atomic force microscopy (AFM). The disassembly of these complexes occurred after they were immersed in ultra-pure water. AFM results showed that the disassembly depended strongly on bridge ions that were used to immobilize the complex onto mica surface, DNA species and ligands. When Mg²⁺ was used as the bridge ion, the DNA–MB complex was completely disassembled because of the weak interactions between Mg²⁺ and DNA's bases or mica surface. Although if Co²⁺ was used as the bridge ion, the disassembly of the DNA–MB complex mainly depended on the species and shape of DNA. For plasmid DNA pBR 322, plasmid DNA pUC 18 and the linear DNA pBR 322/PstI, the degree of disassembly was gradually increased. Whereas if Co(phen)₃³⁺ was chosen as the ligand, the disassembly of the DNA–Co(phen)₃³⁺ complex was almost blocked because Co(phen)₃³⁺ could hardly diffuse into the ultra-pure water. This obtained information may be useful for practical application of the AFM imaging of biological molecules, especially in liquid.     

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.     

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.     

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.     

Cooperativity and intermediate structures of single-stranded DNA binding-assisted RecA-single-stranded DNA complex formation studied by atomic force microscopy

The formation of a complex between RecA protein and single-stranded (ss) DNA was studied systematically by atomic force microscopy (AFM) by varying incubation time and the molecular ratio of RecA protein to single-stranded DNA binding (SSB) protein. New intermediate structures, such as small circular, tangled, and protruded structures in the absence of SSB and sharply turned structures in the presence of SSB, were clearly identified at the early stage of complex formation. These structures have probably resulted from competitive binding of RecA and SSB to DNA. After long incubation, only fully covered RecA-ssDNA and totally RecA-free SSB-ssDNA complexes were present regardless of RecA concentrations. Together with intermediate structures which consisted of only two parts, that is, ssDNA covered by SSB and by RecA proteins, the observation suggested strong neighbor cooperative binding of RecA to ssDNA assisted by SSB.     

Topographic effects on adhesive force mapping of stretched DNA molecules by pulsed-force-mode atomic force microscopy

Adhesive interaction between a tip and a sample surface was examined on a microscopic scale by pulsed-force-mode atomic force microscopy (PFM-AFM). The signal measured by monitoring pull-off force is influenced by various factors such as topography, elasticity, electrostatic charges, and adsorbed water on surfaces. Here, we focus on the topographic effects on the adhesive interaction. To clarify the topographic influence, the adhesive force measurement of a stretched DNA molecule with a smaller radius of curvature than that of a tip was carried out at low relative humidity (RH) with an alkanethiol-modified tip. The experimental conditions such as low RH and the use of the alkanethiol-modified tip were required to minimise the influence of water capillary force on hydrated DNA strands. The hydrophobic modification of a substrate surface was also important to minimise the adsorbed water effect. The DNA molecules were stretched on the substrate surfaces by an immobilisation process called a dynamic molecular combing method. The two-component vapour-phase surface modification with an alkylsilane mixed with a silane derivative containing an amino end group enhanced the DNA adsorption due to the electrostatic interaction. The experimental results for the topographic effects on the adhesive force mapping were reproducible.     

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.     

Improvement of DNA-visualization in dynamic mode atomic force microscopy in air

Near-contact mode atomic force microscopy (AFM) imaging leads to sharper representations of DNA double strands on mica imaged at ambient conditions compared with noncontact mode AFM. Phase shift was used for feedback control yielding height information using a simple model calculation. No contact between tip and sample occurs. Measured DNA widths were up to four times smaller than measured with the same AFM tip in noncontact mode at ambient condition.     

High-voltage nano-oxidation in deionized water and atmospheric environments by atomic force microscopy

This study used atomic force microscopy (AFM), metallic probes with a nanoscale tip, and high-voltage generators to investigate the feasibility of high-voltage nano-oxidation processing in deionized water (DI water) and atmospheric environments. Researchers used a combination of wire-cutting and electrochemical etching to transform a 20-μm-thick stainless steel sheet into a conductive metallic AFM probe with a tip radius of 60 nm, capable of withstanding high voltages. The combination of AFM, high-voltage generators, and nanoscale metallic probes enabled nano-oxidation processing at 200 V in DI water environments, producing oxides up to 66.6 nm in height and 467.03 nm in width. Oxides produced through high-voltage nano-oxidation in atmospheric environments were 117.29 nm in height and 551.28 nm in width, considerably exceeding the dimensions of those produced in DI water. An increase in the applied bias voltage led to an apparent logarithmic increase in the height of the oxide dots in the range of 200-400 V. The performance of the proposed high-voltage nano-oxidation technique was relatively high with seamless integration between the AFM machine and the metallic probe fabricated in this study.     

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.     

Imaging and nano-dissection of tobacco mosaic virus by atomic force microscopy

Tobacco mosaic virus (TMV) has been deposited on freshly cleaved mica substrates. The topography was investigated by contact, non-contact and lateral-force microscopy under ambient conditions in air. The results were in accord with known dimensions of TMV (i.e. 18 nm in diameter and 300 nm in length). However, convolution of tip shape with TMV morphology resulted in an apparent width of 80–140 nm in the lateral plane, a factor of 4–7 greater than the known diameter. Other artefacts - broadening and double images - were observed and ascribed to tip anomalies. High force loadings and slow repetitive scanning resulted in controlled removal of parts of the TMV structure. Accordingly, it was possible to reveal and image the central core channel of the TMV. The precision and resolution of dissection induced by AFM is currently limited by the shape of the tip, having a 40-nm radius of curvature for standard Si₃N₄ tips. It is estimated that sharper tips, with a radius of curvature of less than 10 nm, should be able to resolve, non-destructively, the protein subunits in the non-contact mode, and selectively remove single subunits in the contact mode.