Resolution Atomic Force Microscopy Research Articles

MOCVD growth of N-polar GaN on on-axis sapphire substrate: Impact of AlN nucleation layer on GaN surface hillock density

We report on the impact of growth conditions on surface hillock density of N-polar GaN grown on nominally on-axis (0001) sapphire substrate by metal organic chemical vapor deposition (MOCVD). Large reduction in hillock density was achieved by implementation of an optimized high temperature AlN nucleation layer and use of indium surfactant in GaN overgrowth. A reduction by more than a factor of five in hillock density from 1000 to 170 hillocks/cm−2 was achieved as a result. Crystal quality and surface morphology of the resultant GaN films were characterized by high resolution x-ray diffraction and atomic force microscopy and found to be relatively unaffected by the buffer conditions. It is also shown that the density of smaller surface features is unaffected by AlN buffer conditions.

The mechanism of kaempferol induced apoptosis and inhibited proliferation in human cervical cancer SiHa cell: From macro to nano.

Kaempferol has been identified as a potential cancer therapeutic agent by an increasing amount of evidences. However, the changes in the topography of cell membrane induced by kaempferol at subcellular- or nanometer-level were still unclear. In this work, the topographical changes of cytomembrane in human cervical cancer cell (SiHa) induced by kaempferol, as well as the role of kaempferol in apoptosis induction and its possible mechanisms, were investigated. At the macro level, MTT assays showed that kaempferol inhibited the proliferation of SiHa cells in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that kaempferol could induce SiHa cell apoptosis, mitochondrial membrane potential disruption, and intracellular free calcium elevation. At the micro level, fluorescence imaging by laser scanning confocal microscopy (LSCM) indicated that kaempferol could also destroy the networks of microtubules. Using high resolution atomic force microscopy (AFM), we determined the precise changes of cellular membrane induced by kaempferol at subcellular or nanometer level. The spindle-shaped SiHa cells shrank after kaempferol treatment, with significantly increased cell surface roughness. These data showed structural characterizations of cellular topography in kaempferol-induced SiHa cell apoptosis and might provide novel integrated information from macro to nano level to assess the impact of kaempferol on cancer cells, which might be important for the understanding of the anti-cancer mechanisms of drugs. SCANNING 38:644-653, 2016. © 2016 Wiley Periodicals, Inc.

The Electric Field of CO Tips and Its Relevance for Atomic Force Microscopy.

Metal tips decorated with CO molecules have paved the way for an impressively high resolution in atomic force microscopy (AFM). Although Pauli repulsion and the associated CO tilting play a dominant role at short distances, experiments on polar and metallic systems show that electrostatic interactions are necessary to understand the complex contrast observed and its distance evolution. Attempts to describe those interactions in terms of a single electrostatic dipole replacing the tip have led to contradictory statements about its nature and strength. Here, we solve this puzzle with a comprehensive experimental and theoretical characterization of the AFM contrast on Cl vacancies. Our model, based on density functional theory (DFT) calculations, reproduces the complex evolution of the contrast between both the Na cation and Cl anion sites, and the positively charged vacancy as a function of tip height, and highlights the key contribution of electrostatic interactions for tip-sample distances larger than 500 pm. For smaller separations, Pauli repulsion and the associated CO tilting start to dominate the contrast. The electrostatic field of the CO-metal tip can be represented by the superposition of the fields from the metal tip and the CO molecule. The long-range behavior is defined by the metal tip that contributes the field of a dipole with its positive pole at the apex. At short-range, the CO exhibits an opposite field that prevails. The interplay of these fields, with opposite sign and rather different spatial extension, is crucial to describe the contrast evolution as a function of the tip height.

Amyloid β-Protein Assembly and Alzheimer's Disease: Dodecamers of Aβ42, but Not of Aβ40, Seed Fibril Formation.

Evidence suggests that oligomers of the 42-residue form of the amyloid β-protein (Aβ), Aβ42, play a critical role in the etiology of Alzheimer's disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aβ42 that occur at the earliest stages of aggregation. We observe features that can be attributed to a monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aβ42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 μM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear preprotofibrillar β-sheet structures. The preprotofibrils are a single Aβ42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the preprotofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aβ40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures.

Directed Growth of Polymer Nanorods Using Surface-Initiated Ring-Opening Polymerization of N-Allyl N-Carboxyanhydride.

A stepwise chemistry route was used to prepare arrays of polymer nanostructures of poly(N-allyl glycine) on Si(111) using particle lithography. The nanostructures were used for studying surface reactions with advanced measurements of atomic force microscopy (AFM). In the first step to fabricate the surface platform, isolated nanopores were prepared within a thin film of octadecyltrichlorosilane (OTS). The OTS served as a surface resist, and the areas of nanopores provided multiple, regularly shaped sites for further reaction. An initiator, (3-aminopropyl)triethoxysilane (APTES), was grown selectively inside the nanopores to define sites for polymerization. The initiator attached selectively to the sites of nanopores indicating OTS prevented nonspecific adsorption. Surface-initiated ring-opening polymerization of N-allyl N-carboxyanhydride with APTES produced polymer nanorods on the nanodots of APTES presenting amine functional groups. The surface changes for each step were monitored using high resolution atomic force microscopy (AFM). Slight variations in the height of the poly(N-allyl glycine) nanorods were observed which scale correspondingly to the initial dimensions of nanopores. The distance between adjacent polymer nanorods was controlled by the size of mesoparticle masks used in the experiment. This surface platform has potential application in biotechnology for smart coatings or biosensors.

Atomic force microscopy study of ionomycin-induced degranulation in RBL-2H3 cells.

Mast cell degranulation is the typical anaphylaxis process of mast cells associated with the release of cytokines, eicosanoids and their secretory granules, which play very important roles in the allergic inflammatory response of the human body upon anaphylactogen stimulation. The calcium ionophore ionomycin is widely used as a degranulation induction agent for mast cell degranulation studies. In the present work, ionomycin-induced degranulation of RBL-2H3 basophilic leukemia cell line cells was investigated in vitro by high resolution atomic force microscopy (AFM). Ionomycin, which could increase the intracellular free Ca2+ level and β-Hexosaminidase release, was found to induce the formation of a kind of peculiar vesicles in the cytoplasm area of RBL-2H3 cells. Those vesicles induced by ionomycin would desintegrate to release a larger amount of granules surrounding RBL-2H3 cells by the controlling of F-actin. These results provide the precise morphological information of ionomycin-induced mast cell degranulation at nanoscale, which could benefit our understanding of ionomycin-induced mast cell anaphylaxis model and also validate the applicability of AFM for the detection of allergic inflammatory response in mast cells. SCANNING 38:525-534, 2016. © 2016 Wiley Periodicals, Inc.

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.

Functionalization of Carbon Materials by Reduction of Diazonium Cations Produced in Situ in a Brønstedt Acidic Ionic Liquid

AbstractThe imidazolium‐based acidic ionic liquid 1‐butyl‐3‐methylimidazolium hydrogensulfate, [BMIm][HSO4], is used for the grafting of carbon materials, allowing the nitrobenzene diazonium cations to be simply produced in situ. An appealing self‐limiting and self‐patching process occurs in this viscous ionic liquid. Even for low electrolysis charge consumption, the layers are particularly dense and compact at atomic force microscopy resolution. The grafting method is further extended to carbon nanotubes, following either an electrochemical or a chemical route. A bucky paste is easily obtained from [BMIm][HSO4] and carbon nanotubes that could coat a graphite electrode surface. This simple soft method allows: 1) in situ electrografting of carbon nanotubes in [BMIm][HSO4] and/or 2) easy‐to‐handle electrochemical characterizations of the functionalized carbon nanotubes. The covalent functionalization of single‐walled carbon nanotubes is further assessed by Raman spectroscopy.

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.

Mechanical properties of elastomeric proteins studied by single molecule force spectroscopy

Elastomeric proteins are a special class of proteins with unique mechanical functions. They bear, transduce mechanical forces inside cell, and serve as biomaterials of high elasticities and strengths outside cell. Depending on their functions, the mechanical properties of elastomeric proteins are very diverse. Some of them are of high mechanical stability and the others are of high extensibility and toughness. Although many elastomeric proteins are engineered for the applications in the fields of biomaterials and nanotechnology, the molecular determinant of the mechanical stability remains elusive. In this review, we summarize recent advances in the field of protein mechanics studied by using single molecule force spectroscopy. Force spectroscopy enables people to probe the unfolding properties of protein domains, thus paving the way for building special proteins with characteristic mechanical functions. To begin with, it is necessary to clarify the factors and their relations with the unfolding force, which is deduced based on Bell's expression. It turns out that the unfolding force is proportional to pulling speed when the speed is relatively small, and has a logarithmic relation in the high-speed approximation. After the external determinant of the force probe is clarified, some intrinsic factors are to be discussed. Hydrogen bound and electrostatic force, rather than covalent bond, contribute to the mechanical performances of proteins. Those interactions rely on the topology structures of protein molecules. By changing the structures of proteins, researchers now manage to change the mechanical characteristics of certain proteins. Since single protein is unable to be detected by traditional optic microscope, three devices used to observe and manipulate single protein are introduced in the present paper. These include atomic force microscopy, magnetic tweezers and optical tweezers. Among them, a more detailed explanation of atomic force microscope (AFM) is provided, which briefly describes the basic mechanism and structure of AFM and possible explanation for the formation of force-extension curves. After that, several recent advances for improving the AFM based single molecule force spectroscopy techniques are highlighted. For example, Tom Perkins group [Sullan R M A, Churnside A B, Nguyen D M, Bull M S, Perkins T T 2013 Methods 60 131] has discovered that the gold-stripped tip gives more accurate and reproducible results than a gold-coated one. Matthias Rief group [Schlierf M, Berkemeier F, Rief M 2007 Biophys. J. 93 3989] has managed to increase the resolution of AFM, pushing it in pair with optical tweezers. Hermann Gaub et al. [Otten M, Ott W, Jobst M A, Milles L F, Verdorfer T, Pippig D A, Nash M A, Gaub H E 2014 Nat. Methods 11 1127] combined the microfluidic chip and DNA expression in vitro to increase the yields of interpretable single-molecule interaction traces. Toshio Ando et al. [Ando T, Uchihashi T, Fukuma T 2008 Prog. Surf. Sci. 83 337] have developed methods to increase the imaging speed of AFM. Finally, the rationally designing the mechanical properties of protein-based materials pioneered by Hongbin Li group is highlighted. They have discovered direct relationship between the mechanical properties of individual proteins and those of the protein materials. To sum up, with AFM, scientists now can explore mechanical properties of a wide range of proteins, which enables them to build biomaterials with exceptional mechanical features.

High resolution atomic force microscopy of double-stranded RNA.

Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex.

Electrical Charging Characteristics of Palladium Nanoparticles Synthesized on Tobacco Mosaic Virus Nanotemplate for Organic Memory Device

In this study, an organic memory using palladium nanoparticles (Pd NPs) synthesized on surface of tobacco mosaic virus (TMV) as charge storage elements was demonstrated on metal-pentacene-insulator-silicon (MPIS) structure. The Pd NPs were preferentially composed on precisely spaced thiol functionalities of genetically modified TMV. Laterally dispersed and assembled Pd NPs TMV layer was acted as a platform enabling electrical charging effect for flatband voltage shift (ΔVFB) of memory capacitor derived from hysteresis of capacitance-voltage (C-V) characteristics. The synthesized Pd NPs TMV was confirmed and imaged by high resolution atomic force microscope (AFM) and electrical charging property of the Pd NPs on the TMV was analyzed by electric force microscopy (EFM) and the C-V measurement. The C-V hysteresis behavior was shown in clockwise direction and memory window of ΔVFB in the (+/−) 3 V and (+/−) 35 V range were 0.15 V and 1.14 V, respectively. This study could provide another nanotemplate example of virus application for organic memory device.

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.

Qualitative and Quantitative Analysis of ROS-Mediated Oridonin-Induced Oesophageal Cancer KYSE-150 Cell Apoptosis by Atomic Force Microscopy.

High levels of intracellular reactive oxygen species (ROS) in cells is recognized as one of the major causes of cancer cell apoptosis and has been developed into a promising therapeutic strategy for cancer therapy. However, whether apoptosis associated biophysical properties of cancer cells are related to intracellular ROS functions is still unclear. Here, for the first time, we determined the changes of biophysical properties associated with the ROS-mediated oesophageal cancer KYSE-150 cell apoptosis using high resolution atomic force microscopy (AFM). Oridonin was proved to induce ROS-mediated KYSE-150 cell apoptosis in a dose dependent manner, which could be reversed by N-acetylcysteine (NAC) pretreatment. Based on AFM imaging, the morphological damage and ultrastructural changes of KYSE-150 cells were found to be closely associated with ROS-mediated oridonin-induced KYSE-150 cell apoptosis. The changes of cell stiffness determined by AFM force measurement also demonstrated ROS-dependent changes in oridonin induced KYSE-150 cell apoptosis. Our findings not only provided new insights into the anticancer effects of oridonin, but also highlighted the use of AFM as a qualitative and quantitative nanotool to detect ROS-mediated cancer cell apoptosis based on cell biophysical properties, providing novel information of the roles of ROS in cancer cell apoptosis at nanoscale.

Creating periodic local strain in monolayer graphene with nanopillars patterned by self-assembled block copolymer

A simple and viable method was developed to produce biaxial strain in monolayer graphene on an array of SiO2 nanopillars. The array of SiO2 nanopillars (1 cm2 in area, 80 nm in height, and 40 nm in pitch) was fabricated by employing self-assembled block copolymer through simple dry etching and deposition processes. According to high resolution micro-Raman spectroscopy and atomic force microscopy analyses, 0.9% of maximum biaxial tensile strain and 0.17% of averaged biaxial tensile strain in graphene were created. This technique provides a simple and viable method to form biaxial tensile strain in graphene and offers a practical platform for future studies in graphene strain engineering.

Nucleation and Early Stages of Layer-by-Layer Growth of Metal Organic Frameworks on Surfaces.

High resolution atomic force microscopy (AFM) is used to resolve the evolution of crystallites of a metal organic framework (HKUST-1) grown on Au(111) using a liquid-phase layer-by-layer methodology. The nucleation and faceting of individual crystallites is followed by repeatedly imaging the same submicron region after each cycle of growth and we find that the growing surface is terminated by {111} facets leading to the formation of pyramidal nanostructures for [100] oriented crystallites, and triangular [111] islands with typical lateral dimensions of tens of nanometres. AFM images reveal that crystallites can grow by 5–10 layers in each cycle. The growth rate depends on crystallographic orientation and the morphology of the gold substrate, and we demonstrate that under these conditions the growth is nanocrystalline with a morphology determined by the minimum energy surface.

The ionic liquid assisted green synthesis of hydroxyapatite nanoplates by Moringa oleifera flower extract: A biomimetic approach

This report deals an attempt for a successful green synthesis of hydroxyapatite nanoplates assisted from Moringa oleifera flower extract and 1-butyl-3-methylimidazolium tetrafluoroborate ionic liquid. The prepared nanoplates were calcinated at two different temperatures and the crystallinity, particles size and morphology were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), high resolution transmission electron microscopy (HRTEM) and atomic force microscopy (AFM) techniques. These results proved the crystal growth nucleation of HAp nanoplates at different calcination temperatures. FTIR results were established the interaction of ion precursors with polyphenolic hydroxyl groups from flower extract. The important role of ionic liquid assessment also confirmed the formation of amorphous into crystalline plate like structures.

Characterization of individual molecular adsorption geometries by atomic force microscopy: Cu-TCPP on rutile TiO2 (110)

Functionalized materials consisting of inorganic substrates with organic adsorbates play an increasing role in emerging technologies like molecular electronics or hybrid photovoltaics. For such applications, the adsorption geometry of the molecules under operating conditions, e.g., ambient temperature, is crucial because it influences the electronic properties of the interface, which in turn determine the device performance. So far detailed experimental characterization of adsorbates at room temperature has mainly been done using a combination of complementary methods like photoelectron spectroscopy together with scanning tunneling microscopy. However, this approach is limited to ensembles of adsorbates. In this paper, we show that the characterization of individual molecules at room temperature, comprising the determination of the adsorption configuration and the electrostatic interaction with the surface, can be achieved experimentally by atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). We demonstrate this by identifying two different adsorption configurations of isolated copper(ii) meso-tetra (4-carboxyphenyl) porphyrin (Cu-TCPP) on rutile TiO2 (110) in ultra-high vacuum. The local contact potential difference measured by KPFM indicates an interfacial dipole due to electron transfer from the Cu-TCPP to the TiO2. The experimental results are verified by state-of-the-art first principles calculations. We note that the improvement of the AFM resolution, achieved in this work, is crucial for such accurate calculations. Therefore, high resolution AFM at room temperature is promising for significantly promoting the understanding of molecular adsorption.

Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins.

Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.

Investigation of quercetin-induced HepG2 cell apoptosis-associated cellular biophysical alterations by atomic force microscopy.

Quercetin, a wildly distributed bioflavonoid, has been proved to possess excellent antitumor activity on hepatocellular carcinoma (HCC). In the present study, the biophysical properties of HepG2 cells were qualitatively and quantitatively determined using high resolution atomic force microscopy (AFM) to understand the anticancer effects of quercetin on HCC cells at nanoscale. The results showed that quercetin could induce severe apoptosis in HepG2 cells through arrest of cell cycle and disruption of mitochondria membrane potential. Additionally, the nuclei and F-actin structures of HepG2 cells were destroyed by quercetin treatment as well. AFM morphological data showed some typical apoptotic characterization of HepG2 cells with increased particle size and roughness in the ultrastructure of cell surface upon quercetin treatment. As an important biophysical property of cells, the membrane stiffness of HepG2 cells was further quantified by AFM force measurements, which indicated that HepG2 cells became much stiffer after quercetin treatment. These results collectively suggest that quercetin can be served as a potential therapeutic agent for HCC, which not only extends our understanding of the anticancer effects of quercetin against HCC cells into nanoscale, but also highlights the applications of AFM for the investigation of anticancer drugs.