Mode Atomic Force Microscopy Imaging Research Articles

Error-corrected AFM: a simple and broadly applicable approach for substantially improving AFM image accuracy

Atomic force microscopy (AFM) has become an indispensable tool for imaging the topography and properties of surfaces at the nanoscale. A ubiquitous problem, however, is that optimal accuracy demands smooth surfaces, slow scanning, and expert users, contrary to many AFM applications and practical use patterns. Accordingly, a simple correction to AFM topographic images is implemented, incorporating error signals such as deflection and/or amplitude data that have long been available but quantitatively underexploited. This is demonstrated to substantially improve both height and lateral accuracy for expert users, with a corresponding 3–5 fold decrease in image error. Common image artifacts due to inexperienced AFM use, generally poorly scanned surfaces, or high speed images acquired in as fast as 7 s, are also shown to be effectively rectified, returning results equivalent to standard ‘expert-user’ images. This concept is proven for contact mode AFM, AC-mode, and high speed imaging, as well as property mapping such as phase contrast, with obvious extensions to many specialized AFM variations as well. Conveniently, as this correction procedure is based on either real time or post-processing, it is easily employed for future as well as legacy AFM systems and data. Such error-corrected AFM therefore offers a simple, broadly applicable approach for more accurate, more efficient, and more user-friendly implementation of AFM for nanoscale topography and property mapping.

Non-covalent Immobilization of Desmoplakin Plakin Domain Molecules by Size-Selected Clusters for AFM Imaging

We exploit the immobilization of proteins by size-selected atomic gold clusters to examine the plakin domain of desmoplakin (PD), a human desmosomal protein that is mutated in severe skin diseases and cardiomyopathy, using atomic force microscopy (AFM) in the liquid phase. Contact mode AFM indicates that the PD does not establish a covalent bond size-selected gold nanocluster supported on a graphite substrate, but tapping mode AFM images indicate enhanced weak adsorption. The protein dimensions obtained are compared with predicted values based on electron microscopy, small-angle X-ray scattering (SAXS) and X-ray crystallographic studies.

Nanoscale surface characterization of aqueous copper corrosion: Effects of immersion interval and orthophosphate concentration

Morphology changes for copper surfaces exposed to different water parameters were investigated at the nanoscale with atomic force microscopy (AFM), as influenced by changes in pH and the levels of orthophosphate ions. Synthetic water samples were designed to mimic physiological chemistries for drinking water, both with and without addition of orthophosphate over a pH range 6.5–9. Copper surfaces treated with orthophosphate as a corrosion inhibitor after 6 and 24h were evaluated. Tapping mode AFM images revealed dosing of the water with 6mg/L of orthophosphate was beneficial in retarding the growth of copper by-products. The chemical composition and oxidation state of the surface deposits were characterized with X-ray diffraction (XRD), near edge X-ray absorption fine structure (NEXAFS) spectroscopy and Fourier transform infrared spectroscopy (FTIR).

Vibrational Response of FeNi3 Nanoparticles to the Flux of a Modulated Electromagnetic Field Detected by Contact-Mode Atomic Force Microscopy

A hybrid imaging mode was developed for characterizing samples of magnetic FeNi3 nanoparticles, which combines contact-mode atomic force microscopy (AFM) with magnetic modulation of samples. For conventional magnetic imaging modes of AFM, magnetically coated tips are used directly as a sensor to measure the relatively long-range forces of magnetic samples in a noncontact configuration. For the magnetic sample modulation (MSM) configuration, however, the changes in sample dynamics form the basis for measurements of material properties using contact-mode AFM. Nanoparticles are driven to vibrate in response to an externally applied electromagnetic field, and a nonmagnetic tip is used as a motion sensor for directly mapping the vibration with contact-mode. Intermetallic nanoparticles of FeNi3 were used as a model nanomaterial, synthesized by either conventional oven heating or microwave preparation. By slowly scanning an AFM probe across vibrating nanoparticles, changes in the frequency and amplitude of the s...

Uncovering Ultrastructural Defences in Daphnia magna – An Interdisciplinary Approach to Assess the Predator-Induced Fortification of the Carapace

The development of structural defences, such as the fortification of shells or exoskeletons, is a widespread strategy to reduce predator attack efficiency. In unpredictable environments these defences may be more pronounced in the presence of a predator. The cladoceran Daphnia magna (Crustacea: Branchiopoda: Cladocera) has been shown to develop a bulky morphotype as an effective inducible morphological defence against the predatory tadpole shrimp Triopscancriformis (Crustacea: Branchiopoda: Notostraca). Mediated by kairomones, the daphnids express an increased body length, width and an elongated tail spine. Here we examined whether these large scale morphological defences are accompanied by additional ultrastructural defences, i.e. a fortification of the exoskeleton. We employed atomic force microscopy (AFM) based nanoindentation experiments to assess the cuticle hardness along with tapping mode AFM imaging to visualise the surface morphology for predator exposed and non-predator exposed daphnids. We used semi-thin sections of the carapace to measure the cuticle thickness, and finally, we used fluorescence microscopy to analyse the diameter of the pillars connecting the two carapace layers. We found that D . magna indeed expresses ultrastructural defences against Triops predation. The cuticle in predator exposed individuals is approximately five times harder and two times thicker than in control daphnids. Moreover, the pillar diameter is significantly increased in predator exposed daphnids. These predator-cue induced changes in the carapace architecture should provide effective protection against being crushed by the predator’s mouthparts and may add to the protective effect of bulkiness. This study highlights the potential of interdisciplinary studies to uncover new and relevant aspects even in extensively studied fields of research.

Mechanical mapping of nanobubbles by PeakForce atomic force microscopy

The mechanical behavior of nanobubbles represents their physical essence and has been thought to be closely related to their mysteriously long lifetimes. However, it is difficult to measure the mechanical properties of nanobubbles by conventional atomic force microscopy (AFM). In this paper, nanobubbles were investigated via a novel AFM imaging mode, PeakForce Quantitative Nano-Mechanics (PF-QNM), at the interface of water and highly oriented pyrolytic graphite (HOPG). High resolution images of the nanobubbles in true-contact were achieved by PF-QNM and compared with those obtained by tapping mode AFM (TM-AFM) in the same area. From the force curves simultaneously captured during the PF-QNM imaging processes, the stiffness of the nanobubbles was derived and mapped, ranging usually from 60 to 120 pN nm−1, indicating that the gas–water interface of nanobubbles has similar mechanical properties to those of microbubbles. Interestingly, a size dependence of the stiffness was found and the small nanobubbles had a higher stiffness.

Atomic force microscopy imaging of lipid rafts of human breast cancer cells

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.

Combined Atomic Force Microscopy–Fluorescence Microscopy: Analyzing Exocytosis in Alveolar Type II Cells

Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.

Analysis of time-resolved interaction force mode AFM imaging using active and passive probes

We present an in-depth analysis of time-resolved interaction force (TRIF) mode imaging for atomic force microscopy (AFM). A nonlinear model of an active AFM probe, performing simultaneous topography and material property imaging on samples with varying elasticity and adhesion is implemented in Simulink®. The model is capable of simulating various imaging modes, probe structures, sample material properties, tip-sample interaction force models, and actuation and feedback schemes. For passive AFM cantilevers, the model is verified by comparing results from the literature. As an example of an active probe, the force sensing integrated readout and active tip (FIRAT) probe is used. Simulation results indicate that the active and damped nature of FIRAT provides a significant level of control over the force applied to the sample, minimizing sample indentation and topography error. Active tip control (ATC) preserves constant contact time during force control for stable contact while preventing the loss of material property information such as elasticity and adhesive force. Simulation results are verified by TRIF mode imaging of the samples with both soft and stiff regions.

Intrinsically high-Q dynamic AFM imaging in liquid with a significantly extended needle tip

Atomic force microscope (AFM) probe with a long and rigid needle tip was fabricated and studied for high Q factor dynamic (tapping mode) AFM imaging of samples submersed in liquid. The extended needle tip over a regular commercially available tapping-mode AFM cantilever was sufficiently long to keep the AFM cantilever from submersed in liquid, which significantly minimized the hydrodynamic damping involved in dynamic AFM imaging of samples in liquid. Dynamic AFM imaging of samples in liquid at an intrinsic Q factor of over 100 and an operational frequency of over 200 kHz was demonstrated. The method has the potential to be extended to acquire viscoelastic material properties and provide truly gentle imaging of soft biological samples in physiological environments.

Interactions of nanobubbles with bovine serum albumin and papain films on gold surfaces

Nanobubbles formed on monocrystalline gold/water interface by means of the ethanol-to-water solvent exchange were exposed to the solutions of either bovine serum albumin or papain proteins. Both proteins do not change the position of nanobubbles in water, as observed by in situ tapping mode atomic force microscopy imaging before and after the introduction of the protein. The aqueous environment was subsequently replaced by ethanol. While all nanobubbles were found to dissolve in ethanol in the presence of bovine serum albumin, most of them survived when papain was employed. The protective ability of papain was ascribed to its resistance towards the protein denaturation in aqueous solutions of ethanol. The authors employed in situ atomic force nanolithography to investigate the nanomorphology of the papain/nanobubble assemblies in ethanol.

Localization factor: A new parameter for the quantitative characterization of surface structure with atomic force microscopy (AFM)

In this work we present the possible application of a new parameter called localization factor for the quantitative characterization of surface structures with atomic force microscopy (AFM). For this purpose contact mode AFM images were taken from technologically different polycrystalline gold thin films and were evaluated according to the following parameters: surface roughness ( R a , R RMS ), roughness factor ( f r ) and localization factor. The localization factor was compared with the other surface parameters. We demonstrate that this new parameter can be used to identically characterize these gold thin film surfaces with contact mode AFM in the 1–1000 μm 2 scan range. The mathematical background and possible application fields of the localization factor are also discussed in our paper.

Calcium ions promote formation of amyloid β-peptide (1-40) oligomers causally implicated in neuronal toxicity of Alzheimer's disease.

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1–40) and that of Aβ(1–40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca2+, Aβ(1–40) preferentially formed oligomers similar to those formed by Aβ(1–40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1–40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1–40), that have been implicated in the pathogenesis of AD.

Mechanical Mapping of Single Proteins at Subnanometer Resolution using AFM

The structure and function of proteins are determined by their mechanical stability. The temperature- or B-factors obtained from crystallographic data provide an indirect measure of the order and stability of the protein, together with structural information. However, B-factors from crystallography analysis may be related to the protein packing in the 3D-crystal and are not quantitative. A direct quantification of the mechanical stability of proteins based on atomic force microscopy (AFM) consists in mechanically unfolding individual proteins by pulling from two points. However, its correlation with protein structure requires additional measurements. In this work we apply a novel AFM imaging mode based on force spectroscopy (PeakForce) to simultaneously acquire structural and mechanical information of membrane proteins. We used the well-known protein bacteriorhodopsin from Halobaterium salinarum as a model system, obtaining topographical and stiffness maps with subnanometer resolution. The characteristic trimeric organization of the trigonally packed (a = b = 62.5A; γ= 120°) bacteriorhodopsin was clearly visible with inter-monomer distances of 3.2 nm. Overlay of stiffness and topography maps allowed the investigation of nanomechanical properties at the single molecule level providing a map of the structural stability of bacteriorhodopsin.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Atomic force microscopy of microvillous cell surface dynamics at fixed and living alveolar type II cells

Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca(2+) channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis.

Mechanical and electronic characteristics of scanning probe microscopy probes based on coaxial palladium nanowire/carbon nanotube hybrid structures

The authors present the mechanical and electronic characterization of a novel kind of scanning probe microscopy probe fabricated by the microwave plasma-enhanced chemical vapor deposition growth of carbon nanotubes using a catalytic palladium film deposited only near the apices of commercial tapping mode atomic force microscopy (AFM) cantilevers with the use of a newly developed controlled-area electroplating method. This process is shown to result in the growth of coaxial palladium nanowire/carbon nanotube composite structures (PdNWCNTs). The authors demonstrate that neither the cantilever quality factor nor the cantilever spring constant is significantly degraded by PdNWCNT growth, and show that PdNWCNT probes are adequate for standard tapping mode AFM imaging. Low resistance Ohmic contact between PdNWCNT probes and metal surfaces is demonstrated. Importantly, repeated surface contact and current flow is shown to not damage the PdNWCNTs, indicating that the probes are appropriate for multiprobe conductivity measurements. A brief overview of the fabrication process is also provided.

Chemical Mechanical Polishing of a Ti-Si-N Nanocomposite and AFM Study on Its Nanostructure

Ti-Si-N films were characterized as a nanocomposite consisting of cubic titanium nitride (TiN) nanocrystallites embedded in an amorphous matrix of silicon nitride (Si3N4). Ti-Si-N films have been used as anti-wear coatings due to their mechanical properties and diffusion barriers for copper metallization in semiconductor processing. The surface of Ti-Si-N is so rough that it is hard to characterize its nanostructure with an atomic force microscope (AFM). Therefore, the Ti-Si-N surface should be polished and smoothened to reduce its roughness before an AFM study. In this study, we investigated a chemical mechanical polishing (CMP) process for preparing Ti-Si-N surfaces for an AFM study on the Ti-Si-N nanostructure by using two different types of CMP slurries. Then a characterization of TiN embedded in a Si3N4 matrix was conducted by using a tapping mode AFM and phase shift imaging.

Quantitative mapping of elastic moduli at the nanoscale in phase separated polyurethanes by AFM

The micro phase separated nanoscale morphology of phase separated polyurethanes (PUs) was visualized by atomic force microscopy (AFM) height and phase imaging of smooth surfaces obtained by ultramicrotonomy. PUs were obtained from 4,4′-methylenbis (phenyl isocyanate) (MDI), 1,4-butanediol (BD) and poly(tetrahydrofurane) polyether polyol (PTHF). The segmented polyether PUs with varying stoichiometric ratio of the isocyanate and hydroxyl groups were prepared to investigate the effect of molar mass, as well as the type and number of end-groups on their morphology and mechanical performance.The PU samples studied show characteristic “fingerprint” AFM phase images. Novel dynamic imaging modes of AFM, including HarmoniX material mapping and Peak Force Tapping were used to assess the mechanical performance of phase separated polyurethanes quantitatively as a function of their molecular structure. The values of surface elastic moduli were determined with nanoscale resolution and were in excellent agreement for both AFM modes. While tensile testing provides a bulk average value for the elastic modulus of the elastomers, the novel AFM based elastic moduli mappings introduced enable the study of surface stiffness with nanoscale resolution in a quantitative way.

Atomic force microscopy of biological samples

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate how this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).

Adsorption behavior of partially collapsed polyacrylate coils on mica surfaces: A reciprocal space approach

AbstractIn this article, we investigate tapping mode atomic force microscopy images of intermediate states along the coil to globule transition of sodium polyacrylate coils containing Ca2+ as specifically binding ions. The structural correlations within single adsorbed molecules are established using power spectral density (PSD) curves. The PSD curves of several single molecules are averaged to give the so called 2D form factor so as to obtain information of higher statistical merit. A proper interpretation of the 2D form factor and comparison with form factor analysis of the very same sample solution available through small angle neutron scattering provides an alternative quantification of changes in conformation which a single polyacrylate molecule undergoes as it moves from 3D solution to 2D surface and is inevitably distorted in shape because of sample history. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1553–1561, 2010