Resolution Atomic Force Microscopy Images Research Articles

Are High Resolution Atomic Force Microscopy images proportional to the force gradient or the force maps?

Frequency modulation Atomic Force Microscopy using tips functionalized with carbon monoxide (also known as High Resolution AFM) shows an extraordinary resolution on molecules and it can disclose their inner structure on the frequency shift maps. When operating the microscope at low amplitudes, <1 Å, these frequency shift measurements have been traditionally interpreted as the force gradient between the probe and the sample. Here, we show that, for CO-terminated tips, this interpretation fails even for amplitudes as small as 0.2–0.3 Å. The main reason behind this failure is the very rapid variation in tip-sample interaction upon tip approach associated with the mobility of the CO probe, which deflects to reduce the Pauli repulsion with the sample. Besides, we propose a new and very simple approach for the frequency shift in terms of the force at the closest tip-sample separation. This force approximation reproduces the real frequency maps for amplitudes larger than ∼0.3 Å, independent of CO deflection.

The cyto-linker and scaffolding protein "plectin" mis-localization leads to softening of cancer cells.

Discovering tools to prevent cancer progression requires understanding the fundamental differences between normal and cancer cells. More than a decade ago, atomic force microscopy (AFM) revealed cancer cells' softer body compared to their healthy counterparts. Here, we investigated the mechanism underlying the softening of cancerous cells in comparison with their healthy counterparts based on AFM high resolution stiffness tomography and 3D confocal microscopy. We showed microtubules (MTs) network in invasive ductal carcinoma cell cytoskeleton is basally located and segmented for around 400 nm from the cell periphery. Additionally, the cytoskeleton scaffolding protein plectin exhibits a mis-localization from the cytoplasm to the surface of cells in the carcinoma which justifies the dissociation of the MT network from the cell's cortex. Furthermore, the assessment of MTs' persistence length using a worm-like-chain (WLC) model in high resolution AFM images showed lower persistence length of the single MTs in ductal carcinoma compared to that in the normal state. Overall, these tuned mechanics support the invasive cells to ascertain more flexibility under compressive forces in small deformations. These data provide new insights into the structural origins of cancer aids in progression.

Structure of one-dimensional monolayer Si nanoribbons on Ag(111)

One-dimensional silicon nanoribbons (SiNRs) have the potential for application to future electronic devices because of their compatibility with current silicon-based electronic devices and their theoretical outstanding electronic properties, such as the size-dependent band gap. Here we grew SiNRs by the deposition of Si on a Ag(111) surface. We investigated SiNRs on the Ag(111) surface using a combination of scanning tunneling microscopy (STM), atomic force microscopy (AFM), and density functional theory (DFT) calculations. The atomic resolution STM and AFM images revealed that SiNRs have the same width and align along the equivalent orientations of Ag(111). The main body of SiNRs was found to be symmetric about the long axes of the ribbons, but the terminals of SiNRs break the symmetry. The DFT calculation results revealed a buckled single-layer structure of SiNRs, which is composed of Si hexagonal and tetragonal rings.

Machine learning-based multidomain processing for texture-based image segmentation and analysis

Atomic and molecular resolved atomic force microscopy (AFM) images offer unique insights into materials' properties such as local ordering, molecular orientation, and topological defects, which can be used to pinpoint physical and chemical interactions occurring at the surface. Utilizing machine learning for extracting underlying physical parameters increases the throughput of AFM data processing and eliminates inconsistencies intrinsic to manual image analysis, thus enabling the creation of reliable frameworks for qualitative and quantitative evaluation of experimental data. Here, we present a robust and scalable approach to the segmentation of AFM images based on flexible pre-selected classification criteria. The usage of supervised learning and feature extraction allows us to retain the consideration of specific problem-dependent features (such as types of periodical structures observed in the images and the associated numerical parameters: spacing, orientation, etc.). We highlight the applicability of this approach for the segmentation of molecular resolved AFM images based on the crystal orientation of the observed domains, automated selection of boundaries, and collection of relevant statistics. Overall, we outline a general strategy for machine learning-enabled analysis of nanoscale systems exhibiting periodic order that could be applied to any analytical imaging technique.

Nitrosative stress affects the interaction of integrin alphaIIbbeta3 with its ligands

Binding of integrin alphaIIbbeta3 (αiibβ3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibβ3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibβ3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210–450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibβ3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions.

Primary, Secondary, Tertiary and Quaternary Structure Levels in Linear Polysaccharides: From Random Coil, to Single Helix to Supramolecular Assembly.

Polysaccharides are ubiquitous in nature and represent an essential class of biopolymers with multiple levels of conformation and structural hierarchy. However, a standardized structural nomenclature, as in the case of proteins, is still lacking due to uncertainty on their hierarchical organization. In this work we use carrageenans as model polysaccharides to demonstrate that several structural levels exist and can be unambiguously resolved by statistical analysis on high resolution Atomic Force Microscopy images, supported by spectroscopic, X-ray scattering and rheological techniques. In direct analogy with proteins, we identify primary, secondary, tertiary and quaternary structures. The structure-property relationship induced by monovalent ions for κ-, ι- and the non-gelling control λ-carrageenan is established from the single chain regime to the occurrence of hydrogels at higher concentrations. For κ-carrageenan in the presence of potassium, a disorder-order transition from random coil to single helix is first observed (secondary structure), followed by intrachain supercoiling events (tertiary structure) and macroscopic anisotropic domains which are parts of a network (quaternary structure) with tunable elasticity up to ∼103 Pa. In contrast, κ-carrageenan in the presence of sodium only produces changes in secondary structure without supercoiling events, prior to formation of gels, highlighting the ion-specificity of the process. Loosely intertwined single helices are observed for ι-carrageenan in the presence of sodium and potassium chloride, providing an elastic mesh with many junction zones, while λ-carrageenan does not undergo any structural change. A generality of the observed behavior may be inferred by extending these observations to a distinct class of polysaccharides, the weak carboxylic polyelectrolyte Gellan gum. These results advance our understanding of ion-specific structural changes of polysaccharides and the physical mechanisms responsible for their gelation.

Pore Forming Properties of Alamethicin in Negatively Charged Floating Bilayer Lipid Membranes Supported on Gold Electrodes.

Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.

The conformation and dynamics of P-glycoprotein in a lipid bilayer investigated by atomic force microscopy

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed “small” and “large” protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these “small” and “large” protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 Å AFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the “small” and “large” protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

Unstrained PbSe/CdSe core shell nanostructures for broad band absorber and narrow band IR emitters

The purpose of this work is to establish structural, morphological and optical properties of PbSe quantum dots (QDs) and PbSe/CdSe core/shell nanostructures (C/S NSs) for application as broad band absorber and narrow band emitters. These NSs were synthesized using chemical bath deposition technique. The structural properties were studied using X-ray diffraction technique with Rietveld refinement and transmission electron microscopy (TEM). The surface morphology of synthesized thin film was established using atomic force microscopy (AFM) and scanning electron microscope (SEM). The optical properties were explained using UV–Vis absorption and photoluminescence (PL) spectroscopy with electron–hole recombination process. A comparison of estimated crystallite size from XRD, high resolution AFM and TEM images was resulted in good agreement as ~ 6.2 ± 0.5 and ~ 6.5 ± 0.5 nm for PbSe QDs and PbSe/CdSe C/S NSs respectively.These obtained properties of self-assembled PbSe/CdSe C/S NSs are expected to be useful in the development of broad band absorber and narrowband IR emitters and solar cells.

Direct visualization of alamethicin ion pores formed in a floating phospholipid membrane supported on a gold electrode surface

Unilamellar DMPC/DMPG vesicles in the absence and presence of alamethicin were fused onto the surface of a gold electrode modified with a 1-thio-β-d-glucose self-assembled monolayer. The resulting floating bilayer lipid membranes (fBLMs) were investigated using atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). A corrugated film structure was observed for the pure DMPC/DMPG fBLMs due to surface stress between the tightly packed lipids. These corrugations are removed by the addition of alamethicin suggesting the lipid-peptide interactions alleviate the overall surface stress creating a more uniform bilayer. Both DMPC/DMPG films in the absence and presence of alamethicin had thickness of 5.5 ± 0.9 nm demonstrating that alamethicin has a minimal effect on the overall bilayer thickness. However, a significant decrease in membrane resistivity was observed when alamethicin was inserted into the fBLM indicating that the peptides are forming ion conducting pores. A direct visualization of the alamethicin pores was obtained by molecular resolution AFM images revealing that the pores are not randomly dispersed throughout the bilayer, but instead form hexagonal aggregates. The diameter of an individual pore within the aggregates is equal to 2.3 ± 0.3 nm, which is consistent with the size of a hexameric pore predicted by molecular dynamics simulations. Additionally, the image revealed a broad size distribution of alamethicin aggregates, which explains the origin of multiple conductivity states observed for the incorporation of alamethicin into free standing bilayer lipid membranes.

A comparative study of conventional type II and inverted core–shell nanostructures based on CdSe and ZnS

The objective of this work is to investigate structural, morphological and optical properties of conventional CdSe/ZnS core–shell and inverted ZnS/CdSe core–shell nanostructures for opto-electronic device applications. For this purpose both nanostructures were synthesized using chemical bath deposition technique in thin film form. The structural properties were studied using X-ray diffraction technique with Rietveld refinement and transmission electron microscopy (TEM). The surface morphology of synthesized thin film was illustrated in the form 2D and 3D images using atomic force microscopy (AFM). The optical properties were explained using UV–Vis absorption spectroscopy and photo luminescence (PL) spectroscopy in in situ monitoring process. A comparison of estimated particle size from XRD, high resolution AFM and TEM images was resulted in good agreement as 2.1, 2.4 and 2.1 nm respectively for conventional CdSe/ZnS core–shell and as 2.5, 2.5 and 2.2 nm respectively for inverted ZnS/CdSe core–shell nanostructures.

Erosion of Fe-W model system under normal and oblige D ion irradiation

The dynamic erosion behaviour of iron-tungsten (Fe-W) model films (with 1.5 at% W) resulting from 250eV deuterium (D) irradiation is investigated under well-defined laboratory conditions. For three different impact angles (0°, 45° and 60° with respect to the surface normal) the erosion yield is monitored as a function of incident D fluence using a highly sensitive quartz crystal microbalance technique (QCM). In addition the evolution of the Fe-W film topography and roughness with increasing fluence is observed using an atomic force microscope (AFM). The mass removal rate for Fe-W is found to be comparable to the value of a pure Fe film at low incident fluences but strongly decreases with increasing D fluence. This is consistent with earlier observations of a substantial W enrichment at the surface due to preferential Fe sputtering. The reduction of the mass removal rate is initially more pronounced for irradiation under oblique angles as compared to normal incidence, but the differences vanish for fluences > 2·1023D/m². High resolution AFM images reveal that continued ion irradiation leads to significant surface roughening and (depending on ion impact angle) formation of nanodots or nano-ripples. This may indicate that the W enrichment at the surface due to preferential sputtering of Fe is not exclusively responsible for the observed reduction in erosion with increasing D fluence.

Spatial confinement induces hairpins in nicked circular DNA.

In living cells, DNA is highly confined in space with the help of condensing agents, DNA binding proteins and high levels of supercoiling. Due to challenges associated with experimentally studying DNA under confinement, little is known about the impact of spatial confinement on the local structure of the DNA. Here, we have used well characterized slits of different sizes to collect high resolution atomic force microscopy images of confined circular DNA with the aim of assessing the impact of the spatial confinement on global and local conformational properties of DNA. Our findings, supported by numerical simulations, indicate that confinement imposes a large mechanical stress on the DNA as evidenced by a pronounced anisotropy and tangent–tangent correlation function with respect to non-constrained DNA. For the strongest confinement we observed nanometer sized hairpins and interwound structures associated with the nicked sites in the DNA sequence. Based on these findings, we propose that spatial DNA confinement in vivo can promote the formation of localized defects at mechanically weak sites that could be co-opted for biological regulatory functions.

Immunomodulatory effects of polysaccharide compounds in macrophages revealed by high resolution AFM.

Polysaccharide compounds (PCs), which composed of different kinds of polysaccharides always isolated from different kinds of traditional Chinese medicine, are now attracting more and more attentions due to their strong immunomodulatory activities beyond the corresponding one-component polysaccharides. In this study, we demonstrated for the first time that PCs-1 and PCs-2 had strong immunomodulatory effects on macrophages both in in vitro and in vivo models by atomic force microscopy (AFM). By high resolution AFM imaging, PCs-1 and PCs-2 were found to inhibit LPS induced cell surface particle size and roughness increase in RAW264.7 macrophages, demonstrating the anti-inflammatory effects of PCs-1 and PCs-2 on macrophages. PCs-1 and PCs-2 were also proved to increase the particle size and roughness of resting RAW264.7 macrophages, which suggested that PCs could activate resting RAW264.7 macrophages. And additionally, PCs-1 and PCs-2 were also found to reverse the surface particle size and roughness decrease of peritoneal macrophages isolated from cyclophosphamide induced immunosuppressive mice, suggesting the activation effects of PCs-1 and PCs-2 on immunosuppressive macrophages. These results further enhanced our understanding of macrophage activations by direct imaging of cell surface ultrastructure and also highlighted AFM as a novel nanotool for macrophage detections. And most importantly, these results also indicated the outstanding immunomodulatory effects of PCs on macrophages, which therefore suggested that PCs could be served as a kind of novel immunomodulatory agents that would benefit human health. SCANNING 38:792-801, 2016. © 2016 Wiley Periodicals, Inc.

Submolecular Resolution Imaging of Molecules by Atomic Force Microscopy: The Influence of the Electrostatic Force.

The forces governing the contrast in submolecular resolution imaging of molecules with atomic force microscopy (AFM) have recently become a topic of intense debate. Here, we show that the electrostatic force is essential to understand the contrast in atomically resolved AFM images of polar molecules. Specifically, we image strongly polarized molecules with negatively and positively charged tips. A contrast inversion is observed above the polar groups. By taking into account the electrostatic forces between tip and molecule, the observed contrast differences can be reproduced using a molecular mechanics model. In addition, we analyze the height dependence of the various force components contributing to the high-resolution AFM contrast.

Double-Tip Artefact Removal from Atomic Force Microscopy Images.

The Atomic Force Microscope (AFM) allows the measurement of interactions at interfaces with nanoscale resolution. Imperfections in the shape of the tip often lead to the presence of imaging artefacts such as the blurring and repetition of objects within images. Generally, these artefacts can only be avoided by discarding data and replacing the probe. Under certain circumstances (e.g., rare, high value samples, or extensive chemical/physical tip modification) such an approach is not feasible. Here, we apply a novel deblurring technique, using a Bayesian framework, to yield a reliable estimation of the real surface topography without any prior knowledge of the tip geometry (blind reconstruction). A key contribution is to leverage the significant recently successful body of work in natural image deblurring to solve this problem. We focus specifically on the 'double-tip' effect, where two asperities 1 are present on the tip, each contributing to the image formation mechanism. Finally, we demonstrate that the proposed technique successfully removes the 'double-tip' effect from high resolution AFM images which demonstrate this artefact whilst preserving feature resolution.

High Resolution Imaging Atomic Force Microscope Study of Interactions at the Membrane-Fluid Interface

The cell membrane is essential for all living systems, serving as a barrier between cells and their environment. It is typically composed of a lipid bilayer, containing embedded and/or anchored proteins that mediate different biological function such as energy conversion, signal transduction and solute transport [1]. To elucidate the basic structure of biological membranes it is necessary to make direct experimental observations of the molecular organization of the protein and lipid bilayer under physiologically relevant conditions. Using a bespoke high resolution Atomic Force Microscopy (AFM) it is possible to characterize the structure and function of both native and model lipid membranes with embedded proteins [2].During this study we focus our attention on transmembrane MvP voltage-gated potassium channels embedded in a lipid bilayer, which are activated by changes in transmembrane potential [3]. High resolution AFM images of the membrane channel reveal the predicted tetrameric channel structure of the ion channel. Interestingly, we observed the formation of an asymmetric depression in the supporting lipid membrane surrounding the channel. This may be due to an alteration in the lipid bilayer structure to accommodate the ion channel. Using AFM is possible to investigate the interactions between proteins and membrane- liquid interface [4], [5] aiding our understanding and leading to future therapeutic application.[1] D. J. Muller et al., Nature protocols, vol. 2, no. 9, pp. 2191--2197, 2007.[2] A. Sumino et al., Scientific reports, vol. 3, 2013.[3] A. M. Randich et al., Biochemistry, vol. 53, pp. 1627--1636, 2014.[4] K. H. Sheikh and S. P. Jarvis, Journal of the American Chemical Society, vol. 133, pp. 18296--18303, 2011.[5] U. M. Feber et al., Eur Biophys, vol. 40, pp. 329-338, 2011.

Interfacial Shear Strength of Multilayer Graphene Oxide Films.

Graphene oxide (GO) is considered as one of the most promising layered materials with tunable physical properties and applicability in many important engineering applications. In this work, the interfacial behavior of multilayer GO films was directly investigated via GO-to-GO friction force microscopy, and the interfacial shear strength (ISS) was measured to be 5.3 ± 3.2 MPa. Based on high resolution atomic force microscopy images and the available chemical data, targeted molecular dynamics simulations were performed to evaluate the influence of functional structure, topological defects, and interlayer registry on the shear response of the GO films. Theoretical values for shear strength ranging from 17 to 132 MPa were predicted for the different structures studied, providing upper bounds for the ISS. Computational results also revealed the atomic origins of the stochastic nature of friction measurements. Specifically, the wide scatter in experimental measurements was attributed to variations in functional structure and topological defects within the sliding volume. The findings of this study provide important insight for understanding the significant differences in strength between monolayer and bulk graphene oxide materials and can be useful for engineering topological structures with tunable mechanical properties.

Characterization of the surface charge distribution on kaolinite particles using high resolution atomic force microscopy

Most solid surfaces, in particular clay minerals and rock surfaces, acquire a surface charge upon exposure to an aqueous environment due to adsorption and/or desorption of ionic species. Macroscopic techniques such as titration and electrokinetic measurements are commonly used to determine the surface charge and ζ-potential of these surfaces. However, because of the macroscopic averaging character these techniques cannot do justice to the role of local heterogeneities on the surfaces. In this work, we use dynamic atomic force microscopy (AFM) to determine the distribution of surface charge on the two (gibbsite-like and silica-like) basal planes of kaolinite nanoparticles immersed in aqueous electrolyte with a lateral resolution of approximately 30nm. The surface charge density is extracted from force–distance curves using DLVO theory in combination with surface complexation modeling. While the gibbsite-like and the silica-like facet display on average positive and negative surface charge values as expected, our measurements reveal lateral variations of more than a factor of two on seemingly atomically smooth terraces, even if high resolution AFM images clearly reveal the atomic lattice on the surface. These results suggest that simple surface complexation models of clays that attribute a unique surface chemistry and hence homogeneous surface charge densities to basal planes may miss important aspects of real clay surfaces.

Piezoelectric oscillation sensor based noncontact atomic force microscope for imaging in both ambient and liquid environments

A new design of a noncontact atomic force microscope (AFM) is introduced in this paper, based on a piezoelectric oscillator sensor (PEOS) for ambient and liquid environments. Because of the recent development of quartz technology, the PEOS sensor operates independently from conventional laser alignments. The sensor is based on the length extension resonator, which has high force sensitivity and can deliver high resolution AFM images in ultrahigh vacuum. The oscillator design was tested in different gas compositions and liquids to determine its oscillation stability. The scan performance was investigated in both air and liquid on the topography of an inorganic hard material, graphite. The usability of PEOS for soft organic materials was further proven by imaging biological samples of DNA origami.