Atomic Force Microscopy In Air Research Articles

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment.

Fibrillar collagen-integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril-integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.

Atomic force microscopy measurements and model of DNA bending caused by binding of AraC protein.

Atomic force microscopy (AFM) was used to conduct single-molecule imaging of protein/DNA complexes involved in the regulation of the arabinose operon of Escherichia coli. In the presence of arabinose, the transcription regulatory protein AraC binds to a 38 bp region consisting of the araI1 and araI2 half-sites. The domain positioning of full-length AraC, when bound to DNA, was not previously known. In this study, AraC was combined with 302 and 560 bp DNA and arabinose, deposited on a mica substrate, and imaged with AFM in air. High resolution images of 560 bp DNA, where bound protein was visible, showed that AraC induces a bend in the DNA with an angle 60°± 12° with a median of 55°. These results are consistent with earlier gel electrophoresis measurements that measured the DNA bend angle based on migration rates. By using known domain structures of AraC, geometric constraints, and contacts determined from biochemical experiments, we developed a model of the tertiary and quaternary structure of DNA-bound AraC in the presence of arabinose. The DNA bend angle predicted by the model is in agreement with the measurement values. We discuss the results in view of other regulatory proteins that cause DNA bending and formation of the open complex to initiate transcription.

Tip-Substrate Shear Interaction in Quartz Tuning Fork-Based Atomic Force Microscopy in Air

Tip-Substrate Shear Interaction in Quartz Tuning Fork-Based Atomic Force Microscopy in Air

Comparison of Different Excitation Schemes in Bimodal Atomic Force Microscopy in Air and Liquid Environments

Bimodal amplitude modulation atomic force microscopy (AM-AFM) is widely used in nanoscale topography and compositional contrast imaging for various materials. In this work, we use computational simulation to compare the dynamic behaviors of AFM cantilevers in three commonly used excitation schemes in bimodal AM-AFM, i.e., the cantilever base excitation, the cantilever end excitation, and the uniform excitation along the length of the cantilever, in both air and liquid environments. The amplitude and phase spectroscopy curves and the frequency responses acquired from the three excitation schemes are compared and discussed. The results would be useful in guiding the selection of excitation methods and the optimization of imaging conditions.

Evaluation of the Topographical Surface Changes of Silicon Wafers after Annealing and Plasma Cleaning

The morphological stability of silicon single crystal wafers was investigated, after performing cleaning surface treatments based on moderate temperature annealing and plasma sputtering. The wafer surfaces were measured by Tapping mode atomic force microscopy in air, before and after the different treatments. The 3D images were segmented by watershed algorithm identifying the local peaks, and the stereometric parameters were extracted thereof. The analysis of variance allowed to better assess the statistically significant differences. All the resulting quantities were critically discussed. It appeared that the different cleaning treatments affected differently the surface morphology changes occurring between pristine and treated surfaces, making them distinguishable in these terms. The presented combination of measurement technique and analyzing protocol potentially allows one to assess the structural differences of the surfaces of interest, when assumptions are made about the physical origin of the emerging topographical features. In the present case, if no etching is assumed, it appears that all cleaning protocols actually worsen the surface quality. The effect of these morphological differences on the functional properties of the surface should be ascertained independently.

Imaging aquatic animal cells and associated pathogens by atomic force microscopy in air.

Atomic force microscopy (AFM) is a sophisticated imaging tool with nanoscale resolution that is widely used in structural biology, cell biology, and material science, among other fields. However, to date it has rarely been applied to the study of aquatic animals, especially on one of the main cultured species, shrimp. One reason for this is that no shrimp cell line established until now, primary cell is fragile and difficult to be studied under AFM. In this study, we used AFM to image three different types of biological material from shrimp (Litopenaeus vannamei) in air, including hemocytes and two associated pathogens. Without obvious deformations when the cells were imaged in air and in the case for the haemocytes and the cells were fixed as well. The result suggests hydrophobic glass coverslips are a suitable substrate for adhesion of these samples. The method described here can be applied to the preparation of other fragile biological samples from aquatic animals for high-resolution analyses of host-pathogen interactions and other basic physiological processes.

Stability and Catalytic Performance of Reconstructed Fe3O4(001) and Fe3O4(110) Surfaces during Oxygen Evolution Reaction

Earth-abundant oxides are promising candidates as effective and low-cost catalysts for the oxygen evolution reaction (OER) in alkaline media, which remains one of the bottlenecks in electrolysis and artificial photosynthesis. A fundamental understanding of the atomic-scale reaction mechanism during OER could drive further progress, but a stable model system has yet to be provided. Here we show that Fe3O4 single crystal surfaces, prepared in ultrahigh vacuum (UHV) are stable in alkaline electrolytein the range pH 7-14 and under OER conditions in 1 M NaOH. Fe3O4(001) and Fe3O4(110) surfaces were studied with X-ray photoelectron spectroscopy, low-energy electron diffraction, and scanning tunneling microscopy in UHV, and atomic force microscopy in air. Fe3O4(110) is found to be more reactive for oxidative water splitting than (001)-oriented magnetite samples. Magnetite is electrically conductive, and the structure and properties of its major facets are well understood in UHV. With these newly obtained results, we propose magnetite (Fe3O4) as a promising model system for further mechanistic studies of electrochemical reactions in alkaline media and under highly oxidizing conditions.

Atomic-Force Microscopy Probe-Activated Morphological Transformations in a Nanophase Copper Wetting Layer on Silicon

The phase transition of the nanophase Cu2Si wetting layer on a Si(001) substrate to more stable Cu silicide has been selectively activated by atomic-force microscopy in air. The transition is accompanied by an increase in the lateral size and height of grains and a decrease in their density. The effect that has been identified can be used to form ordered nanoisland ensembles and in nanolithography.

Морфологические превращения в нанофазном смачивающем слое меди на кремнии, активированные воздействием зонда атомно-силового микроскопа

AbstractThe phase transition of the nanophase Cu_2Si wetting layer on a Si(001) substrate to more stable Cu silicide has been selectively activated by atomic-force microscopy in air. The transition is accompanied by an increase in the lateral size and height of grains and a decrease in their density. The effect that has been identified can be used to form ordered nanoisland ensembles and in nanolithography.

Physical-mechanical image of the cell surface on the base of AFM data in contact mode

Physical and mechanical properties of the cell surface are well-known markers of a cell state. The complex of the parameters characterizing the cell surface properties, such as the elastic modulus (E), the parameters of adhesive (Fa), and friction (Ff) forces can be measured using atomic force microscope (AFM) in a contact mode and form namely the physical-mechanical image of the cell surface that is a fundamental element of the cell mechanical phenotype. The paper aims at forming the physical-mechanical images of the surface of two types of glutaraldehyde-fixed cancerous cells (human epithelial cells of larynx carcinoma, HEp-2c cells, and breast adenocarcinoma, MCF-7 cells) based on the data obtained by AFM in air and revealing the basic difference between them. The average values of friction, elastic and adhesive forces, and the roughness of lateral force maps, as well as dependence of the fractal dimension of lateral force maps on Z-scale factor have been studied. We have revealed that the response of microscale areas of the HEp-2c cell surface having numerous microvilli to external mechanical forces is less expressed and more homogeneous in comparison with the response of MCF-7 cell surface.

(Invited) High-Resolution Scanning Electrochemical Microscopy of Biological and Artificial Nanopores

In this presentation, we discuss applications of scanning electrochemical microscopy (SECM) to high-resolution imaging of biological and artificial nanopores. SECM imaging enables us to obtain unprecedented information about the kinetics, pathways, and mechanism of molecular transport through single nanopores. We characterize nanopipet electrodes as nanoscale SECM probes by transmission electron microscopy to verify their small diameters of ~30 nm. The resultant high spatial resolution of our SECM instrument is quantitatively demonstrated by imaging ion transport through the single nanopores of a thin solid silicon membrane. The nanoscale SECM approach thus established is applied to image a biological nanopore, the nuclear pore complex (NPC), in aqueous solution, which eliminates artifacts that are caused when the NPC is imaged by electron microscopy in vacuum and atomic force microscopy in air. Our SECM study of single NPCs is significant, because it will led to a greater mechanistic understanding of molecular transport through the NPC, which plays crucial roles in the gene expression regulation and gene delivery and is linked to many human diseases and therapeutics of genetic disorders.

Key factors of scanning a plant virus with AFM in air and aqueous solution.

For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about ∼30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude (A0 ) and the amplitude set-point (A) are also assessed here. While high A0 prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 Nm-1 ) and high A0 (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point (A/A0 = 0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm. Microsc. Res. Tech. 80:18-29, 2017. © 2016 Wiley Periodicals, Inc.

Length-extension resonator as a force sensor for high-resolution frequency-modulation atomic force microscopy in air

Frequency-modulation atomic force microscopy has turned into a well-established method to obtain atomic resolution on flat surfaces, but is often limited to ultra-high vacuum conditions and cryogenic temperatures. Measurements under ambient conditions are influenced by variations of the dew point and thin water layers present on practically every surface, complicating stable imaging with high resolution. We demonstrate high-resolution imaging in air using a length-extension resonator operating at small amplitudes. An additional slow feedback compensates for changes in the free resonance frequency, allowing stable imaging over a long period of time with changing environmental conditions.

Protein Adsorption on Chemically Modified Block Copolymer Nanodomains: Influence of Charge and Flow.

Understanding the interactions of biomacromolecules with nanoengineered surfaces is vital for assessing material biocompatibility. This study focuses on the dynamics of protein adsorption on nanopatterned block copolymers (BCPs). Poly(styrene)-block-poly(1,2-butadiene) BCPs functionalized with an acid, amine, amide, or captopril moieties were processed to produce nanopatterned films. These films were characterized using water contact angle measurements and atomic force microscopy in air and liquid to determine how the modification process affected. wettability and swelling. Protein adsorption experiments were conducted under static and dynamic conditions via a quartz crystal microbalance with dissipation. Proteins of various size, charge, and stability were investigated to determine whether their physical characteristics affected adsorption. Significantly decreased contact angles were caused by selective swelling of modified BCP domains. The results indicate that nanopatterned chemistry and experimental conditions strongly impact adsorption dynamics. Depending on the structural stability of the protein, polyelectrolyte surfaces significantly increased adsorption over controls. Further analysis suggested that protein stability may correlate with dissipation versus frequency plots.

Growth of single gold nanofilaments at the apex of conductive atomic force microscope tips.

This paper describes a fast and one-step technique to grow single gold filaments at the apex of commercial conductive AFM tips. It is implemented with an atomic force microscope in air with a high relative humidity at room temperature and is based on a bias-assisted electro-reduction of gold ions directly at the tip apex. The technique requires only ad hoc substrates made of a mesoporous silica layer loaded with gold salt deposited on a conductive electrode. It leads to the growth, at the tip apex, of filaments whose length can be monitored and controlled during the growth between tens and hundreds of nanometers and whose radius of curvature can be as low as 3 nm. Made of polycrystalline gold nanostructures, the filaments are chemically and mechanically stable and conductive.

Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA–SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA–SWNT hybrids was 1.1±0.3nm and 2.4±0.6nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4±3.1nm and 10.0±4.5nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA–SWNT hybrids. The morphology of the SSB–ssDNA–SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB–ssDNA–SWNT hybrids showed much larger variance than the ssDNA–SWNT hybrids.

Humidity Dependence of Tribochemical Wear of Monocrystalline Silicon.

The nanowear tests of monocrystalline silicon against a SiO2 microsphere were performed using an atomic force microscope in air as a function of relative humidity (RH=0%-90%) and in liquid water at a contact pressure of about 1.20 GPa. The experimental results indicated that RH played an important role in the nanowear of the Si/SiO2 interface. In dry air, a hillock-like wear scar with a height of ∼0.4 nm was formed on the silicon surface. However, with the increase of RH, the wear depth on the silicon surface first increased to a maximum value of ∼14 nm at 50% RH and then decreased below the detection limit at RH above 85% or in water. The transmission electron microscopy analysis showed that the serious wear on the silicon surface at low and medium RHs occurred without subsurface damage, indicating that the wear was due to tribochemical reactions between the Si substrate and the SiO2 counter surface, rather than mechanical damages. The RH dependence of the tribochemical wear could be explained with a model involving the formation of "Si-O-Si" chemical bonds (bridges) between two solid surfaces. The suppression of tribochemical wear at high RHs or in liquid water might be attributed to the fact that the thickness of the interfacial water layer is thick enough to prevent the solid surfaces from making chemical bridges. The results may help us understand the nanowear mechanism of silicon that is an important material for dynamic microelectromechanical systems.

Multi-eigenmode control for high material contrast in bimodal and higher harmonic atomic force microscopy

High speed imaging and mapping of nanomechanical properties in atomic force microscopy (AFM) allows the observation and characterization of dynamic sample processes. Recent developments involve several cantilever frequencies in a multifrequency approach. One method actuates the first eigenmode for topography imaging and records the excited higher harmonics to map nanomechanical properties of the sample. To enhance the higher frequencies’ response two or more eigenmodes are actuated simultaneously, where the higher eigenmode(s) are used to quantify the nanomechanics. In this paper, we combine each imaging methodology with a novel control approach. It modifies the Q factor and resonance frequency of each eigenmode independently to enhance the force sensitivity and imaging bandwidth. It allows us to satisfy the different requirements for the first and higher eigenmode. The presented compensator is compatible with existing AFMs and can be simply attached with minimal modifications. Different samples are used to demonstrate the improvement in nanomechanical contrast mapping and imaging speed of tapping mode AFM in air. The experiments indicate most enhanced nanomechanical contrast with low Q factors of the first and high Q factors of the higher eigenmode. In this scenario, the cantilever topography imaging rate can also be easily improved by a factor of 10.

Imaging and force measurement of LDL and HDL by AFM in air and liquid

The size and biomechanical properties of lipoproteins are tightly correlated with their structures/functions. While atomic force microscopy (AFM) has been used to image lipoproteins the force measurement of these nano-sized particles is missing. We detected that the sizes of LDL and HDL in liquid are close to the commonly known values. The Young’s modulus of LDL or HDL is ∼0.4GPa which is similar to that of some viral capsids or nanovesicles but greatly larger than that of various liposomes. The adhesive force of LDL or HDL is small (∼200pN). The comparison of AFM detection in air and liquid was also performed which is currently lacking. Our data may provide useful information for better understanding and AFM detection of lipoproteins.

Robust deposition of lambda DNA on mica for imaging by AFM in air.

Long DNA molecules remain difficult to image by atomic force microscopy (AFM) because of their tendency to entanglement and spontaneous formation of networks. We present a comparison of two different DNA deposition methods operating at room temperature and humidity conditions, aimed at reproducible imaging of isolated and relaxed λ DNA conformations by AFM in air. We first demonstrate that a standard deposition procedure, consisting in adsorption of DNA in the presence of divalent cations followed by washing and air-drying steps, yields a coexistence of different types of λ DNA networks with a only a few isolated DNA chains. In contrast, deposition using a spin-coating-based technique results in reproducible coverage of a significant fraction of the substrate area by isolated and relaxed λ DNA molecules, with the added benefit of a reduction in the effect of a residual layer that normally embeds DNA strands and leads to an apparent DNA height closer to the expected value. Furthermore, we show that deposition by spin-coating is also well-suited to visualize DNA-protein complexes. These results indicate that spin-coating is a simple, powerful alternative for reproducible sample preparation for AFM imaging.