Unique insight into protein-DNA interactions from single molecule atomic force microscopy

Tetrapods have the unique property of self-aligning on a flat surface with one arm pointing in the direction perpendicular to the surface plane. The positioning of single tetrapods on the flattened tip of commercial AFM probes (see image) is reported. These tetrapod-functionalized AFM probes are used to image the topography of different samples revealing excellent spatial and vertical resolution. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Dynamic imaging of single DNA–protein interactions using atomic force microscopy

We present a fast method for computing simulated Atomic Force Microscope (AFM) image scans (including tip artifacts). The basic insight is that the array of depth values in the depth buffer of a graphics system is analogous to the height field making up an AFM image, and thus the ability of graphics hardware to compute the depth of many pixels in parallel can be used to radically speed up the AFM imaging simulation. We also present a method for reconstructing better approximations to the true shape from AFM images distorted by tip artifacts. These algorithms are implemented using operations from 3D grayscale mathematical morphology operations of dilation and erosion.

Application of image alignment and time averaging methods in AFM detection for single DNA molecules

Nanotubes, particularly single-walled carbon nanotubes (SWCNTs), represent an important class of materials with valuable electrical, mechanical, and thermal properties. However, accurate characterization of nanotube network morphologies remains a significant challenge. We present a deep learning-based approach for extracting nanotube morphologies from atomic force microscopy (AFM) images utilizing an image-to-image (I2I) translation framework based on cycleGAN with a specialized loss function that learns to transform AFM images containing nanotubes to images of pure substrates. By subtracting these translated substrate images from the original AFM images, we effectively isolated nanotube morphologies even on substrates with roughness exceeding the nanotube diameter. We validate our approach through physics-based simulation studies and application to roll-to-roll printed carbon nanotube transistors on flexible polymeric substrates.Our method outperforms tranditional image processing and supervised learning models in sensitivity and accuracy for CNT network extraction. This improved characterization capability provides useful insights for optimizing the fabrication processes of CNT-TFTs, supporting their development for flexible electronic applications. The methodology extendsbeyond carbon nanotubes to other nanomaterial-based electronic devices, with future work aimed at expanding the model's generalization and integrating with real-time AFM imaging.

Ab initio periodic Hartree-Fock calculations with the full potential and minimum basis set are applied to interpretation of scanning tunneling microscope (STM) and atomic force microscope (AFM) images on 1TVTe2. Our results show that the simulated STM image shows asymmetry while the simulated AFM image shows the circular electron densities at the bright spots without asymmetry of electron density to agree with the experimental AFM image. The bright spots of both the STM and AFM images of VTe2 are associated with the surface Te atoms, while the patterns of bright spots of STM and AFM images are different.

In this thesis, two inversion-based feedforward control approaches have been developed and implemented to high-speed large-range atomic force microscope (AFM) imaging and nanofabrication, respectively. High-speed large-range AFM imaging and nanofabrication are needed in many areas and have attracted great interests. Challenges, however, must be overcome because in high-speed largerange AFM operation, large positioning error of the AFM probe relative to the sample can be generated due to the adverse effects of the hardware. The nonlinear hysteresis and the vibrational dynamics effects of the piezotube actuator must be addressed during the high-speed lateral scanning of the AFM imaging over large imaging size. In addition, precision positioning of the AFM probe in the vertical direction is even more challenging (than the lateral scanning) because the desired trajectory (i.e., the sample topography profile) is unknown in general, and the probe positioning is also effected by and sensitive to the probe-sample interaction. Finally, in AFM multi-axis nanofabrication, additional positioning errors are induced by the large cross-axis dynamics coupling effect. The large positioning errors generated during high-speed, large-range fabrication will lead to large defects in the fabricated structures or devices. In the thesis, firstly, to compensate for the adverse effects generated during high-speed large-range AFM imaging, an integrated approach is proposed, which combines the enhanced inversion-based iterative control technique (EIIC) for lateral x-y axis scanning with a dual-stage piezoactuator for the vertical z-axis positioning. The main contribution of this integrated approach is the combination of an advanced control algorithm with an advanced hardware platform. This approach is demonstrated in experiments by imaging a large-size (50 μm) calibration sample at high speed (50 Hz scan rate). Then secondly, to achieve high-speed large-range multi-axis AFM nanofabrication, a recently developed model-less inversion-based iterative control technique (MIIC) is utilized to overcome the adverse effects involved in high-speed large-range multi-axis AFM nanofabrication. By using this advanced control technique,

Visualization of DNA–protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3′-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA–protein interactions by AFM.

The importance of the atomic force microscope (AFM), in nano-technology, demands understanding of factors affecting AFM performance and image quality. Through a simulation study, the impact of scanning parameters on AFM images acquired in contact-mode has been investigated. The performance and limitations of commercial AFM controllers was also discussed. Based on the generated images, it is seen that high speed scans may require a large force set-point to maintain probe-sample contact at the expense of higher surface deformation, contact area, and friction force. Hence, reducing resolution and increasing image distortion. The sensitivity of AFM images to scan and controller parameters, invites the development of a systematic procedure for parameter selection for a given sample and operating environment. This would greatly reduce the current guess work in parameter selection and improve image repeatability. Currently, commercial control systems do limit the capabilities of the AFM in terms of speed and data repeatability. A good controller should be able to compensate for the large uncertainty due to different cantilevers and samples that may be used, nonlinearities, and system resonances. In addition, it should provide a tight tolerance on sample surface tracking both during transient and at steady state operation. This would enable robust high-speed operation and allow small or negative force set-point to be used.

We describe in detail how atomic force microscopy (AFM) images can be routinely achieved with macroscopic silicon-based chips integrating mesoscopic tips, paving the way for the development of new near field devices combining AFM imaging with any kind of functionality integrated on a chip. The chips have been glued at the end of the free prong of 100 kHz quartz tuning forks mounted in Qplus configuration. Numerical simulations by modal analysis have been carried out to clarify the nature of the vibration modes observed in the experimental spectra. It is shown that two low frequency modes can be used to drive the system and scan the surface with a great stability in amplitude modulation as well as in frequency modulation AFM under ultrahigh vacuum. The AFM capabilities are demonstrated through a series of examples including phase and dissipation contrast imaging, force spectroscopy measurements, and investigations of soft samples in weak interaction with the substrate. The lateral resolution with the tips grown by focused ion beam deposition already matches the one achieved in standard amplitude modulation mode AFM experiments.

Quantitative determination of isomorphous substitutions on clay mineral surfaces through AFM imaging: A case of mica

Electrochemical scanning tunneling microscopy (ESTM) and atomic force microscopy (AFM) images of hydrogen-terminated Si(111) in several solutions have been studied to evaluate the performance of the AFM method. In dilute H2SO4 solution, the overall features of AFM images were similar to those of ESTM images, suggesting that both images reflect the topography of the bare H-Si(111) surface. However, AFM images in ultrapure water and in dilute NaOH solution were successfully obtained, while ESTM images were not. These results indicate that AFM is a powerful method for studying topographic change of Si in solutions during the wet cleaning process.

The surfaces of commensurate layered tellurides NbGe{sub 2/5}Te{sub 2}, NbGe{sub 1/3}Te{sub 2}, and NbSi{sub 1/2}Te{sub 2} were examined by scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The observed atomic-resolution STM and AFM images were analyzed in terms of the partial and total electron density plots of their surfaces calculated by the extended Hueckel tight-binding electronic band structure method. The STM and AFM images of these phases are characterized by a pseudohexagonal arrangement of spots representing the surface Te atoms, and the surface unit-cell parameters of these commensurate MA{sub x}Te{sub 2} phases are well reproduced by STM and AFM. The atomic size patterns of the AFM and STM images differ in their relative contrasts from those expected on the basis of the surface Te-atom corrugations. This discrepancy is explained if the surface Te-atom sheets undergo a slight height reconstruction, most likely because the short interlayer Te{center_dot}{center_dot}{center_dot}Te contacts present in the bulk crystals are truncated at the surface. 12 refs., 13 figs.

Surface texture is an important issue to realize the nature (crest and trough) of surfaces. Atomic force microscopy (AFM) image is a key analysis for surface topography. However, in nano-scale, the nature (i.e., deflection or crack) as well as quantification (i.e., height or depth) of deposited layers is essential information for material scientist. In this paper, a gradient-based K-means algorithm is used to differentiate the layered surfaces depending on their color contrast of as-obtained from AFM images. A transformation using wavelet decomposition is initiated to extract the information about deflection or crack on the material surfaces from the same images. Z-axis depth analysis from wavelet coefficients provides information about the crack present in the material. Using the above method corresponding surface information for the material is obtained. In addition, the Gaussian filter is applied to remove the unwanted lines, which occurred during AFM scanning. Few known samples are taken as input, and validity of the above approaches is shown.

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.