Tapping-mode Atomic Force Microscopy Imaging Research Articles

Investigation of temperature induced mechanical changes in supported bilayers by variants of tapping mode atomic force microscopy.

Tapping mode atomic force microscopy (AFM) is an invaluable technique for examining topographical features of biological materials in solution, and there has been a growing interest in developing techniques to provide further compositional contrast and information concerning surface mechanical properties. Phase shifts, cantilever response at higher harmonic frequencies of the drive, and time-resolved tip/sample force reconstruction have all been shown to provide additional compositional contrast of surfaces, as compared to basic tapping mode AFM imaging. This study aimed to demonstrate the relative ability of these different imaging techniques to detect temperature induced changes in the elastic modulus of supported total brain lipid extract (TBLE) bilayer patches on mica. To aid in direct comparison between the different imaging techniques, all required data was obtained simultaneously while capturing traditional tapping mode AFM topography images. While all of the techniques were able to provide compositional contrast consistent with known temperature-induced changes in the bilayer patch, interpretation of the resulting contrast was not always straightforward. Phase imaging suffered from contrast inversion. Individual harmonics responded in a variety of ways to the temperature-induced changes in elastic modulus of the bilayer. The maximum tapping force (or peak force) associated with imaging the bilayer correctly reflected the changes in elastic modulus of the lipid bilayer. Importantly, as the required data can be obtained simultaneously, combining these different imaging techniques can lead to a more complete understanding of a sample's mechanical features.

Challenges in imaging of soft layers and structures at solid surfaces using atomic force microscopy

Imaging of soft, deformable fluid layers and structures, such as submicron droplets and bubbles or two-dimensional fluid patches, at solid interfaces with an atomic force microscopy (AFM) is a very challenging task. In this article, the authors describe the most common imaging AFM modes and advances in tapping mode AFM imaging of soft systems, as well as discuss the most common artifacts occurring in such experiments during imaging of soft and deformable features at solid interfaces. In general, for rigid and deformable systems, images recorded with the AFM are always a convolution of the tip geometry and the shape of the features being imaged. With deformable samples, the size and shape of the imaged features can additionally be affected by the imaging parameters such as set-point or gains. In addition, AFM images can be easily misprocessed with image enhancement software, resulting in an image that does not represent real features at the surface is created. This review will highlight the necessary procedures to ensure successful and accurate image acquisition in soft features in AFM.

Novel Reciprocal Self-Sensing Techniques for Tapping-Mode Atomic Force Microscopy

We evaluate two novel reciprocal self-sensing methods for tapping-mode atomic force microscopy (TM-AFM) utilizing charge measurement and charge actuation, respectively. A microcantilever, which can be batch fabricated through a standard microelectromechanical system (MEMS) process, is coated with a single piezoelectric layer and simultaneously used for actuation and deflection sensing. The setup enables the elimination of the optical beam deflection technique which is commonly used to measure the cantilever oscillation amplitude. The voltage to charge and charge to voltage transfer functions reveal a high amount of capacitive feedthrough which degrades the dynamic range of the sensors significantly. A feedforward control technique is employed to cancel the feedthrough and increase the dynamic range from less than 1 dB to approximately 30 dB. Experiments show that the conditioned self-sensing schemes achieve an excellent signal-to-noise ratio and can therefore be used to provide the feedback signal for TM-AFM imaging.

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.

Investigation of Temperature Induced Mechanical Changes in Supported Bilayers by Reconstructed Atomic Force Microscopy Tapping Forces

Membrane mechanics, such as rigidity, adhesion, and fluidity are known to play an important role in the regulation of many biological and cellular processes. These properties can be drastically affected by changes in temperature; increasing temperature can enhance membrane permeability and susceptibility whereas decreasing temperature is known to restrict membrane movement and the intake of essential nutrients. Temperature also plays a key role in determining the phase of a membrane; evidence of nanoscale rearrangement of lipids into raft-like domains has been found at physiological temperatures. Rafts are thought to be a site of importance for signal transduction and membrane protein trafficking. There is even a growing association between rafts and the development of neurodegenerative diseases. Thus, it is evident that temperature has a critical role in the maintenance of membrane biology. In this work, patches of total brain lipid extract bilayer containing 30% exogenous cholesterol were formed in phosphate buffered saline and examined using atomic force microscopy while the temperature was systematically increased from 28-40°C. Tapping mode atomic force microscopy (TMAFM) and scanning probe acceleration microscopy (SPAM) were used to obtain topographical and mechanical information of the sample, respectively. SPAM is relatively new technique that offers the advantage of quickly obtaining mechanical information, while maintaining nanoscale spatial imaging resolution, due to its basis in TMAFM. The principle of SPAM is that the noisy deflection signal is captured during TMAFM imaging and reconstructed to reveal the time-resolved forces between the cantilever tip and the sample surface. These tip/sample forces can be correlated with surface properties such as rigidity and adhesion. With SPAM it is also possible to construct harmonic images, thus providing further insights on subtle surface characteristics.

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.

Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM

We report on the use of three different atomic force spectroscopy modalities to determine the nanomechanical properties of amyloid fibrils of the human α-synuclein protein. α-Synuclein forms fibrillar nanostructures of approximately 10 nm diameter and lengths ranging from 100 nm to several microns, which have been associated with Parkinson's disease. Atomic force microscopy (AFM) has been used to image the morphology of these protein fibrils deposited on a flat surface. For nanomechanical measurements, we used single-point nanoindentation, in which the AFM tip as the indenter is moved vertically to the fibril surface and back while the force is being recorded. We also used two recently developed AFM surface property mapping techniques: Harmonic force microscopy (HarmoniX) and Peakforce QNM. These modalities allow extraction of mechanical parameters of the surface with a lateral resolution and speed comparable to tapping-mode AFM imaging. Based on this phenomenological study, the elastic moduli of the α-synuclein fibrils determined using these three different modalities are within the range 1.3-2.1 GPa. We discuss the relative merits of these three methods for the determination of the elastic properties of protein fibrils, particularly considering the differences and difficulties of each method.

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.

Numerical analysis of dynamic force spectroscopy using a dual-oscillator sensor

A spectral analysis method was recently introduced by Stark et al. [Proc. Natl. Acad. Sci. U.S.A. 99, 8473 (2002)] and implemented by Sahin et al. [Nat. Nanotechnol. 2, 507 (2007)], which is capable of performing simultaneous tapping-mode atomic force microscopy imaging and force spectroscopy. Here the authors report on numerical dual-oscillator simulations that are applied in combination with Fourier data processing software to explore optimized spectroscopy parameters for characterizing polymer and semiconductor samples with the above mentioned spectral analysis method. They also describe various imaging artifacts observed in the simulations, expected limitations of the dual-oscillator model used, and trends of the effect of the most relevant imaging and data processing variables on the quality of the acquired force curves.

Direct Patterning of Intrinsically Electron Beam Sensitive Polymer Brushes

The fabrication of patterned polymer brushes has attracted considerable attention as these structures can be exploited in devices on the nano- and microscale. Patterning of polymer brushes is typically a complex, multistep process. We report the direct patterning of poly(methyl methacrylate) (PMMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(isobutyl methacrylate) (PIBMA), poly(neopentyl methacrylate) (PNPMA), and poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) brushes in a single step by electron beam (e-beam) lithography, to obtain nanopatterned polymer brush surfaces. PMMA, PHEMA, PIBMA, PNPMA, and PTFEMA brushes were grown on silicon substrates via surface-initiated atom transfer radical polymerization. Surface analysis techniques including ellipsometry, contact angle goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) were used to characterize the thickness, hydrophilicity, roughness, and chemical composition of the polymer brushes. Tapping-mode AFM imaging confirmed the successful electron beam patterning of these brushes. Using this direct patterning method, highly resolved nanostructured polymer brush patterns down to 50 nm lines were obtained. This direct patterning of brushes eliminates the need for complex lithographic schemes. The sensitivity of these polymer brushes toward direct patterning with e-beam was studied and compared. The sensitivity curves indicate that the structure of the e-beam degradable methacrylate polymer has a significant effect on the sensitivity of the polymer brush toward e-beam patterning. In particular, the effect of the chemical functionality at the beta-position to the carbonyl group on the polymer brush sensitivity toward direct patterning was studied using groups of varying size and polarity.

Development of a Directly Patterned Low-Surface-Energy Polymer Brush in Supercritical Carbon Dioxide

Carbon dioxide (CO2) is a sustainable solvent because it is nonflammable, exhibits a relatively low toxicity, and is naturally abundant. As a selective, nonpolar solvent, supercritical CO2 (scCO2) is an ideal fit for the development of low-surface-energy polymers. The development of directly patterned poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) brushes in scCO2 was investigated. PTFEMA, in particular, was selected over other fluorinated polymers because of its very high electron-beam (e-beam) sensitivity. PTFEMA brushes were grown on silicon substrates via controlled surface-initiated atom-transfer radical polymerization of TFEMA. Surface analysis techniques including ellipsometry, contact-angle goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy were used to characterize the thickness, hydrophilicity, roughness, and chemical composition of the polymer brushes. PTFEMA brushes were directly patterned in a single step using e-beam lithography and were processed in an environmentally benign scCO2 solvent. Tapping-mode AFM imaging confirmed the successful e-beam patterning and development of these brushes. The sensitivity of PTFEMA brushes toward direct patterning with the e-beam, followed by scCO2 development, was studied and compared to development in tetrahydrofuran solvent. Using this direct-patterning method, followed by dry development in scCO2, highly resolved nanostructured polymer brush lines down to 78 nm could be prepared. This method can be generalized to prepare fluorinated low-surface-energy polymer brush surfaces in a single step for various applications.

A combined atomic force microscopy imaging and docking study to investigate the complex between p53 DNA binding domain and Azurin

The tumor suppressor p53 interacts with the redox copper protein Azurin (AZ) forming a complex which is of some relevance in biomedicine and cancer therapy. To obtain information on the spatial organization of this complex when it is immobilized on a substrate, we have used tapping mode-atomic force microscopy (TM-AFM) imaging combined with computational docking. The vertical dimension and the bearing volume of the DNA binding domain (DBD) of p53, anchored to functionalized gold substrate through exposed lysine residues, alone and after deposing AZ, have been measured by TM-AFM. By a computational docking approach, a three-dimensional model for the DBD of p53, before and after addition of AZ, have been predicted. Then we have calculated the possible arrangements of these biomolecular systems on gold substrate by finding a good agreement with the related experimental distribution of the height. The potentiality of the approach combining TM-AFM imaging and computational docking for the study of biomolecular complexes immobilized on substrates is briefly discussed.

Controlling Spontaneous Emission of CdSe Nanoparticles Dispersed in Electrospun Fibers of Polycarbonate Urethane

Luminescent fibrous composite films consisting of submicrometer diameter fibers were prepared by electrospinning of segmented polycarbonate urethane (PCU) in dimethyl formamide and tri-n-octylphosphine oxide (TOPO)-capped CdSe nanocrystals (5 nm in diameter) in toluene. Using a pair of conductive electrodes separated with an air gap, we successfully produced randomly deposited and uniaxially aligned electrospun fibers. The surface structure of the electrospun fibers was studied using atomic force microscopy (AFM) and was compared to the corresponding film prepared by casting. In cast film, tapping mode AFM imaging suggests that hard urethane segments organize into rodlike morphology dispersed in soft polycarbonate. When PCU/CdSe dispersions were subjected to electrospinning, copolymer domains were forced to arrange into lamella along the fiber axis due to elongational flow and high stretching. Molecular orientation in the domains of the composite fibers was confirmed by polarized infrared spectroscopy. We demonstrated that formation of the oriented domains by electrospinning develops a hierarchical structure, which consequently modifies spectral properties because new multiple sharp lines appeared in the photoluminescence (PL) spectra of the fibers. In contrast to randomly deposited fibers, the PL intensity of uniaxially aligned fibers was found to be angle dependent. We propose that the elongated internal structure within the fibers controls the spontaneous emission of CdSe nanoparticles dispersed throughout the electrospun mat. A discussion on the nature of the controlled spontaneous emission is provided.

Electrochromic WO3 Films: Nanotechnology Experiments in Instrumental Analysis and Physical Chemistry Laboratories

This experiment teaches students the methodology of investigating novel properties of materials using new instrumental techniques: atomic force microscopy (AFM), electrochemical quartz crystal nanobalance (EQCN), voltammetric techniques (linear potential scan and chronoamperometry), and light reflectance measurements. The unique capabilities of each of these techniques are utilized in a combined effort to control and monitor synthesis of electrochromic nanostructured films and then to investigate and analyze film properties. The experiments are designed to demonstrate the penetration of chemical species into solids and its practical implications. It is shown that the intercalation of ions into solid materials through solid-state diffusion is sufficiently fast, even at room temperature, that it can be recorded in real-time by nanogravimetry (EQCN), voltammetric methods, and light reflectance. The high performance of electrochromic films is achieved owing to the formation of nanostructured WO3 with nanoparticles of 20–30 nm in size. The diameter of nanoparticles can be controlled by changing deposition potential and is examined by tapping-mode AFM imaging. The subject of the experiment is of high practical importance for studies of properties of new materials discovered recently in various fields of nanotechnology. The interests in electrochromic materials stem from applications in digital displays and smart windows. At present, in many laboratories in the world, extensive research studies are under way to develop new materials for such applications. The proposed experiment introduces a new concept of intercalation and also shows how to control nanoparticle deposition and how to measure nanomolar quantities of H+ ions inserted in WO3 nanoparticles. In addition, students learn modern instrumental techniques.

Kinetics of CO2 nanobubble formation at the solid/water interface

The kinetics of adsorption of CO(2) molecules dissolved in aqueous solution onto a hydrophobised silica surface were investigated using a quartz crystal microbalance (QCM). The results of this investigation were compared with those obtained earlier from tapping mode atomic force microscopy (TMAFM) under the same experimental conditions (J. Yang, J. Duan, D. Fornasiero, J. Ralston, J. Phys. Chem. B., 2003, 107(25), 6139-6147; ref. 1). The QCM results represent the early stage of CO(2) gas adsorption (<20 min), before CO(2) gas bubbles adsorbed on the surface can be directly observed by TMAFM. The QCM results confirmed our observation from TMAFM imaging: that CO(2) gas molecules present in solution only adsorb on silica when its surface is hydrophobic. More importantly, the results showed that gas adsorption/bubble growth undergoes two consecutive kinetic processes: a slow and a fast adsorption process.

Electrochemically Controlled Formation and Growth of Hydrogen Nanobubbles

Electrogenerated microscale bubbles that are confined at the electrode surface have already been extensively studied because of their significant influence on electrochemistry. In contrast, as far as we know, whether nanoscale bubbles exist on the electrode surface has not been experimentally confirmed yet. Here, we report the observation of electrochemically controlled formation and growth of hydrogen nanobubbles on bare highly oriented pyrolytic graphite (HOPG) surface via in-situ tapping mode atomic force microscopy (TMAFM). By using TMAFM imaging, we observed that electrochemically generated hydrogen gas led to the formation of nanobubbles at the HOPG surface. We then employed a combination of techniques, including phase imaging, ex-situ degassing, and tip perturbation, to confirm the gas origin of such observed nanobubbles. We further demonstrated that the formation and growth of nanobubbles could be well controlled by tuning either the applied voltage or the reaction time. Remarkably, we could also monitor the evolution process of nanobubbles, that is, formation, growth, coalescence, as well as the eventual release of merged microbubbles from the HOPG surface.

Structural and nanoindentation studies of stem cell-based tissue-engineered bone

Stem cell-based gene therapy and tissue engineering have been shown to be an efficient method for the regeneration of critical-sized bone defects. Despite being an area of active research over the last decade, no knowledge of the intrinsic ultrastructural and nanomechanical properties of such bone tissue exists. In this study, we report the nanomechanical properties of engineered bone tissue derived from genetically modified mesenchymal stem cells (MSCs) overexpressing the rhBMP2 gene, grown in vivo in the thigh muscle of immunocompetent mice for 4 weeks, compared to femoral bone adjacent to the transplantation site. The two types of bone had similar mineral contents (61 and 65 wt% for engineered and femoral bone, respectively), overall microstructures showing lacunae and canaliculi (both measured by back-scattered electron microscopy), chemical compositions (measured by energy dispersive X-ray analysis), and nanoscale topographical morphologies (measured by tapping-mode atomic force microscopy imaging or TMAFM). Nanoindentation experiments revealed that the small length scale mechanical properties were statistically different with the femoral bone (indented parallel to the bone long axis) being stiffer and harder (apparent elastic modulus, E∼27.3±10.5 GPa and hardness, H∼1.0±0.7 GPa) than the genetically engineered bone ( E∼19.8±5.6 GPa, H∼0.9±0.4 GPa). TMAFM imaging showed clear residual indents characteristic of viscoelastic plastic deformation for both types of bone. However, fine differences in the residual indent area (smaller for the engineered bone), pile up (smaller for the engineered bone), and fracture mechanisms (microcracks for the engineered bone) were observed with the genetically engineered bone behaving more brittle than the femoral control.

Characterization of elastomeric blends by atomic force microscopy

AbstractThe bulk mechanical properties of a blend of elastomers are found to depend on the micro and nano scale morphology of the phases of the materials in the blend. In this study, we examine the phase morphology of blends of incompatible elastomers using Atomic Force Microscopy (AFM). Specifically, nanoindentation and Tapping Mode AFM (TMAFM) imaging techniques are used as experimental tools for mapping the composition of unfilled elastomeric blends. Depending on the composition of the blend, either co‐continuous or discontinuous domain/matrix morphology is observed. To identify the different components in bromobutyl (BIIR)/natural rubber (NR) blends, nanoscale indentation measurements were made on the observed phase‐separated regions. Results from force mode AFM and mechanical measurements of bulk NR and BIIR are used to assist in the interpretation of the TMAFM results for the BIIR/NR blends. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 492–503, 2006

Single-Walled Carbon Nanotube AFM Probes: Optimal Imaging Resolution of Nanoclusters and Biomolecules in Ambient and Fluid Environments

Carbon nanotubes are potentially ideal atomic force microscopy (AFM) probes due to their well-defined geometry, robust mechanical properties, and in the case of single-walled nanotubes (SWNTs), diameters approaching the size of small organic molecules. Here we elucidate fundamental factors that determine AFM imaging resolution using a combination of experiments with well-characterized SWNT probes and numerical simulations. Tapping mode AFM imaging on gold nanoclusters and DNA molecules using SWNT probes shows that the tip-induced broadening reaches the geometrical limit (i.e., the diameter of the SWNT tips) in the repulsive imaging regime, and the broadening effect becomes greater than the geometrical limit in the attractive imaging regime due to long-range attractive forces. Adjusting the damping enables imaging of isolated DNA molecules with a resolution defined by the tip diameter without disrupting the sample.

Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly- l-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM–FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM–FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM–FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.