Piezo-thermal probe array for high throughput applications

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Characterisation of electrical resistance for CMC materials up to 2000 K

We report images from atomic force microscopy (AFM) of a heated metallic surface performed under an ambient environment. The stainless steel surface was heated up to 121C, which is limited only by the capacitive heating element used (100W). We observed that the topography of the surface changes dramatically. It is found that at high temperatures, the surface appears flat compared with the presence of many round bumps observed on the same sample at room temperature when the ambient humidity is high. To the best of our knowledge, this is the first report on ambient AFM performed on a metallic surface above 100C.Most AFM systems adopt moving sample stages [1]. In this case, it is difficult to implement heavy heating heating elements and a heat reservoir block. For this experiment, the TAK3 from AT Corporation [2] is used. The sample stage is kept stationary and the heating element (the tip of a soldering iron) is fixed firmly on one side of a surface of the block using a screw.

We present a procedure that allows a reliable determination of the elastic (Young’s) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever’s spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

The ability of biomolecules to associate into various structural configurations has a substantial impact on human physiology. The synthesis of protein polypeptide chains using the information encoded by DNA is mediated through the use of regulatory proteins, known as transcription factors. Some transcription factors perform function by inducing local curvature in deoxyribonucleic acid (DNA) strands, the mechanisms of which are not entirely known. An important architectural protein, eleven zinc finger CTCF (11 ZF CTCF) is involved in genome organization and hypothesized to mediate DNA loop formation. Direct evidence for these CTCF-induced DNA loops has yet to be observed. In this thesis, the effect of 11 ZF CTCF on DNA morphology is examined using atomic force microscopy, a powerful technique for visualizing biomolecules with nanometer resolution. The presence of CTCF is revealed to induce a variety of morphologies deviating from the relaxed state of control DNA samples, including compact circular complexes, meshes, and networks. Images reveal quasi-circular DNA/CTCF complexes consistent with a single DNA molecule twice wrapped around the protein. The structures of DNA and proteins are highly important for operations in the cell. Structural irregularities may lead to a variety of issues, including more than twenty human pathologies resulting from aberrant protein misfolding into amyloid aggregates of elongated fibrils. Insulin deficiency and resistance characterizing type 2 diabetes often requires administration of insulin. Injectable and inhalable delivery methods have been documented to result in the deposition of amyloid fibrils. Oligomers, soluble multiprotein assemblies, are believed to play an important role in this process. Insulin aggregation under physiological conditions is not well understood and oligomers have not yet been fully characterized. In this thesis, in vitro insulin aggregation at acidic and neutral pH is explored using a variety of techniques, including kinetic thioflavin T fluorescence, circular dichroism spectroscopy, atomic force and electron microscopy imaging. The size distribution of insulin oligomers at different assembly stages is characterized through covalent cross-linking and gel electrophoresis. Results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, with heptamers formed in 10[micrometre] insulin. These findings suggest that oligomers may be on- and off- pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, required to induce insulin aggregation at neutral pH, increases fibril formation rate and fibrillar mass by an order of magnitude each. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.

Lateral inhomogeneities of modern solar cells demand direct electrical imaging with nanometer resolution. We show that atomic force microscopy (AFM)-based electrical techniques provide unique junction characterizations, giving a two-dimensional determination of junction locations. Two AFM-based techniques, scanning capacitance microscopy/spectroscopy (SCM/SCS) and scanning Kelvin probe force microscopy (SKPFM), were significantly improved and applied to the junction characterizations of multicrystalline silicon (mc-Si) cells. The SCS spectra were taken pixel by pixel by precisely controlling the tip positions in the junction area. The spectra reveal distinctive features that depend closely on the position relative to the electrical junction, which allows us to indentify the electrical junction location. In addition, SKPFM directly probes the built-in potential over the junction area modified by the surface band bending, which allows us to deduce the metallurgical junction location by identifying a peak of the electric field. Our results demonstrate resolutions of 10-40 nm, depending on the techniques (SCS or SKPFM). These direct electrical measurements with nanometer resolution and intrinsic two-dimensional capability are well suited for investigating the junction distribution of solar cells with lateral inhomogeneities.

Chapter 1 - Atomic Force Microscopy as a Nanoanalytical Tool

Probing is increasingly utilized as a technique for the characterization of the local electrical properties of an integrated circuit, as well as the isolation of defects. Test structures and/or SRAM arrays were examined with the various probing modes available with a probing system. Test structures were examined using Electron Beam Absorbed Current (EBAC), Resistive Contrast Imaging (RCI), Electron Beam Induced Current, (EBIC), and EBIRCH (Electron Beam Induced Resistance CHange). The results demonstrate the utility of using each in an SRAM yield management scenario. EBAC is able to provide information about basic connectivity. RCI allows for the isolation of resistive spots along a conductor. EBIC provides for the imaging of depletion zones between PW and NW, even in a planar view. EBIRCH, being driven by two different mechanisms (thermal coefficient of resistivity and Seebeck effect) is able to provide two different kinds of analyses, depending on the conditions. EBIRCH precisely isolated which of a few fins in a multi-fin device are responsible for a short and showed the thermal relations between the elements of a pulldown device in an SRAM. The techniques together provide multiple forms of process feedback when used as part of an integrated yield management program involving analysis of via chains, SRAM parallel array test structures, and SRAMs.

High-Speed AFM Reveals the Dynamics of Single Biomolecules at the Nanometer Scale

Atomic force microscopy has been successfully used to examine a wide range of cellular and biomolecular structures and interactions. The application of atomic force microscopy in the analysis of organs and tissues, however, has been limited. In this study, we present a new method for high-resolution atomic force microscopy imaging of compact bone tissue. We performed atomic force microscopy imaging on demineralized compact bone from bovine tibia to obtain structural information about the bone matrix and the lacunar-canalicular network. Knowledge of the dimensions and distributions of the network allows quantitative analysis of the microfluidics of bone tissue. Results from our study show that (1) the canalicular distribution and dimensions are homogenous in transverse, radial and longitudinal orientations; (2) the lamellae of an osteon consist of alternating high and low bands; (3) the canaliculi follow the contour of lamellar bands and (4) globular structures cover much of the bone matrix, including canalicular walls. Our work demonstrates that atomic force microscopy studies of thin-section tissue samples can provide structural details at nanometre resolution.

Cells are complex, viscoelastic bodies. Their mechanical properties are defined by the arrangement of semiflexible cytoskeletal fibers, their crosslinking, and the active remodeling of the cytoskeletal network. Atomic force microscopy (AFM) is an often‐used technique for the study of cell mechanics, enabling time‐ and frequency‐dependent measurements with nanometer resolution. Cells exhibit time‐dependent deformation when stress is applied. In this work, we have investigated the stress relaxation of HeLa cells when subjected to a constant strain. We have varied the applied force (1, 2, 4, and 8 nN) and pause time (1, 10, and 60 s) to check for common assumptions for the use of models of linear viscoelasticity. Then, we have applied three models (standard linear solid, five element Maxwell, power law rheology) to study their suitability to fit the datasets. We show that the five element Maxwell model captures the stress relaxation response the best while still retaining a low number of free variables. This work serves as an introduction and guide when performing stress relaxation experiments on soft matter using AFM.Research HighlightsCells exhibit linear viscoelastic properties when subjected to stress relaxation measurements at the studied different forces and times.The stress relaxation is best described by a five element Maxwell model.All three used models capture a softening and fluidization of cells when disrupting actin filaments.

Shales are composed of minerals and organic matter, whose individual properties are essential to determining the rock’s macroscopical deformation and strength. Scanning electron microscopy combined with electron energy dispersive spectroscopy (EDS) has been extensively used to evaluate composition, while peak-force atomic force microscopy (AFM) has been used on the determination of elastic modulus with nanometric resolution. Still, there is a need for tools to conduct an in-depth study of the minerals’ tribomechanical properties. Atomic force microscopy is a tool that can contribute to these studies, as it can simultaneously measure the tribomechanical properties and identify the phases. In this work, we propose using atomic force microscopy and energy dispersive spectroscopy to identify the shale components and to measure the in situ tribomechanical properties from the different phases. Friction images between the atomic force microscopy tip and the surface were acquired as a function of load. Minerals and organic matter were later identified by colocalized energy dispersive spectroscopy mapping. Then, the frictional characteristics of the major shale constituents were obtained by adjusting the Derjaguin-Muller-Toporov model to the selected components. Moreover, the identification of the different phases was performed. The results show that friction at the nanometer scale was observed to be higher for organic matter than for any other shale constituent, while shear strength was observed to be higher for quartz and lower for organic matter. These characteristics were used to differentiate shale constituents. It is shown that a careful comparison of friction can be used to differentiate the sulfite pyrite, tectosilicates (quartz, andesine, and albite), phyllosilicate biotite, and organic matter. The presented methodology gives novel information on friction properties in the nanoscale that are comparable to available centimetric characterization techniques contributing to the understanding of rock strength.

Underwater air retention of superhydrophobic hierarchically structured surfaces is of increasing interest for technical applications. Persistent air layers (the Salvinia effect) are known from biological species, for example, the floating fern Salvinia or the backswimmer Notonecta. The use of this concept opens up new possibilities for biomimetic technical applications in the fields of drag reduction, antifouling, anticorrosion and under water sensing. Current knowledge regarding the shape of the air–water interface is insufficient, although it plays a crucial role with regards to stability in terms of diffusion and dynamic conditions. Optical methods for imaging the interface have been limited to the micrometer regime. In this work, we utilized a nondynamic and nondestructive atomic force microscopy (AFM) method to image the interface of submerged superhydrophobic structures with nanometer resolution. Up to now, only the interfaces of nanobubbles (acting almost like solids) have been characterized by AFM at these dimensions. In this study, we show for the first time that it is possible to image the air–water interface of submerged hierarchically structured (micro-pillars) surfaces by AFM in contact mode. By scanning with zero resulting force applied, we were able to determine the shape of the interface and thereby the depth of the water penetrating into the underlying structures. This approach is complemented by a second method: the interface was scanned with different applied force loads and the height for zero force was determined by linear regression. These methods open new possibilities for the investigation of air-retaining surfaces, specifically in terms of measuring contact area and in comparing different coatings, and thus will lead to the development of new applications.

The measurement of intermolecular forces at the liquid-solid interface is key to many studies of electrochemistry, wetting, catalysis, biochemistry, and mechanobiology. The atomic force microscope (AFM) is unique in its ability to measure and map these forces with nanometer resolution using the oscillating sharp tip of an AFM cantilever. These surface forces are only measured by observing the changes they induce in the dynamics of the resonant AFM probe. However, AFM cantilever dynamics at this interface can be significantly different when compared to air/vacuum environments due to the nature of nanoscale forces at the interface and the low-quality factors in liquids. In this work, we study the nonlinear dynamics of magnetically excited AFM microcantilevers on graphite and mica immersed in deionized water, high-concentration buffers, and methanol. By combining theory and experiments, a wealth of nonlinear dynamical phenomena such as superharmonic resonance, hysteretic jumps, and multimodal interactions are demonstrated and their dependence on hydration/solvation forces is clarified. These results are expected to aid ongoing efforts to link liquid-solid interface properties to cantilever dynamics and lead to accurate interpretation of data from experiments.

Integration of confocal and atomic force microscopy images