Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy (AFM)

We present the use of atomic force microscopy (AFM) to visualize and quantify the dynamics of epithelial cell junction interactions under physiological and pathophysiological conditions at the nanoscale. Desmosomal junctions are critical cellular adhesion components within epithelial tissues and blistering skin diseases such as Pemphigus are the result in the disruption of these components. However, these structures are complex and mechanically inhomogeneous, making them difficult to study. The mechanisms of autoantibody mediated keratinocyte disassembly remain largely unknown. Here, we have used AFM technology to image and measure the mechanical properties of living skin epithelial cells in culture. We demonstrate that force measurement data can distinguish cells cultured with and without autoantibody treatment. Our demonstration of the use of AFM for in situ imaging and elasticity measurements at the local, or tissue level opens potential new avenues for the investigation of disease mechanisms and monitoring of therapeutic strategies in blistering skin diseases.     

Partial validation of cross flow ultrafiltration by atomic force microscopy

Atomic force microscopy was used to evaluate a cross flow ultrafiltration (CFUF) system. The CFUF system was used for the size fractionation of natural colloidal material from freshwaters. Analysis of the images of bulk water, permeates, and retentates shows the primary materials observed were near-spherical structures with height dimensions up to approximately 12 nm. The number of colloidal particles (per unit area) on the mica surfaces derived from the retentates increased by a factor of 2 between a concentration factor (cf) of 1 and of 20. Colloidal densities of nanoparticles were approximately 2 orders of magnitude lower in the permeate compared to the retentate, indicating a good size fractionation. As the cf value increased from 1 to 20, the percentage of <1-nm material decreased substantially and the percentage of >1-nm material increased substantially in the retentates. Line transects along a surface and surface roughness values show good agreement with the above results. Results suggest the size fractionation is good but not perfect and high cf values produce a better size fractionation, although some retention of small material is always observed. High cf values are recommended.     

Monitoring DNA immobilization and hybridization on surfaces by atomic force microscopy force measurements

DNA immobilization and hybridization was carried out on Au substrates that were modified with mercaptopropanoic acid and then treated with aluminum(III) solution. The positively charged AI(III) film can be used to immobilize both ds-DNA and ss-DNA. Atomic force microscopy (AFM) was used to monitor the process by force measurements between a negatively charged silica tip and the substrates while immersed in dilute electrolyte. Surface hybridization of ss-DNA produces an increase in the surface charge and surface potential of the substrates, which is reflected by the increasing repulsive force as determined from AFM force-separation curves. A single-base mismatch was detectable in surface hybridization. The AFM force measuring technique was also employed to investigate the interaction of Ru(phen)3(2+) with ss-DNA and ds-DNA. The force measurement results showed that there is a small interaction between Ru(phen)3(2+) and ss-DNA, which was ascribed to the electrostatic binding of Ru(phen)3(2+) to the ss-DNA surface. For ds-DNA, there is a strong interaction which is believed to be due to the association or intercalation of Ru(phen)3(2+) with ds-DNA.     

Uncertainty quantification in nanomechanical measurements using the atomic force microscope

Quantifying uncertainty in measured properties of nanomaterials is a prerequisite for the manufacture of reliable nanoengineered materials and products. Yet, rigorous uncertainty quantification (UQ) is rarely applied for material property measurements with the atomic force microscope (AFM), a widely used instrument that can measure properties at nanometer scale resolution of both inorganic and biological surfaces and nanomaterials. We present a framework to ascribe uncertainty to local nanomechanical properties of any nanoparticle or surface measured with the AFM by taking into account the main uncertainty sources inherent in such measurements. We demonstrate the framework by quantifying uncertainty in AFM-based measurements of the transverse elastic modulus of cellulose nanocrystals (CNCs), an abundant, plant-derived nanomaterial whose mechanical properties are comparable to Kevlar fibers. For a single, isolated CNC the transverse elastic modulus was found to have a mean of 8.1?GPa and a 95% confidence interval of 2.7-20?GPa. A key result is that multiple replicates of force-distance curves do not sample the important sources of uncertainty, which are systematic in nature. The dominant source of uncertainty is the nondimensional photodiode sensitivity calibration rather than the cantilever stiffness or Z-piezo calibrations. The results underscore the great need for, and open a path towards, quantifying and minimizing uncertainty in AFM-based material property measurements of nanoparticles, nanostructured surfaces, thin films, polymers and biomaterials.     

Nanoindentation and atomic force microscopy measurements on reactively sputtered TiN coatings

Titanium nitride (TiN) coatings were deposited by d.c. reactive magnetron sputtering process. The films were deposited on silicon (111) substrates at various process conditions, e.g. substrate bias voltage (V_B) and nitrogen partial pressure. Mechanical properties of the coatings were investigated by a nanoindentation technique. Force vs displacement curves generated during loading and unloading of a Berkovich diamond indenter were used to determine the hardness (H) and Young’s modulus (Y) of the films. Detailed investigations on the role of substrate bias and nitrogen partial pressure on the mechanical properties of the coatings are presented in this paper. Considerable improvement in the hardness was observed when negative bias voltage was increased from 100–250 V. Films deposited at |V _B| = 250 V exhibited hardness as high as 3300 kg/mm². This increase in hardness has been attributed to ion bombardment during the deposition. The ion bombardment considerably affects the microstructure of the coatings. Atomic force microscopy (AFM) of the coatings revealed fine-grained morphology for the films prepared at higher substrate bias voltage. The hardness of the coatings was found to increase with a decrease in nitrogen partial pressure.     

Quantitative force and dissipation measurements in liquids using piezo-excited atomic force microscopy: a unifying theory

The use of a piezoelectric element (acoustic excitation) to vibrate the base of microcantilevers is a popular method for dynamic atomic force microscopy. In air or vacuum, the base motion is so small (relative to tip motion) that it can be neglected. However, in liquid environments the base motion can be large and cannot be neglected. Yet it cannot be directly observed in most AFMs. Therefore, in liquids, quantitative force and energy dissipation spectroscopy with acoustic AFM relies on theoretical formulae and models to estimate the magnitude of the base motion. However, such formulae can be inaccurate due to several effects. For example, a significant component of the piezo excitation does not mechanically excite the cantilever but rather transmits acoustic waves through the surrounding liquid, which in turn indirectly excites the cantilever. Moreover, resonances of the piezo, chip and holder can obscure the true cantilever dynamics even in well-designed liquid cells. Although some groups have tried to overcome these limitations (either by theory modification or better design of piezos and liquid cells), it is generally accepted that acoustic excitation is unsuitable for quantitative force and dissipation spectroscopy in liquids. In this paper the authors present a careful study of the base motion and excitation forces and propose a method by which quantitative analysis is in fact possible, thus opening this popular method for quantitative force and dissipation spectroscopy using dynamic AFM in liquids. This method is validated by experiments in water on mica using a scanning laser Doppler vibrometer, which can measure the actual base motion. Finally, the method is demonstrated by using small-amplitude dynamic AFM to extract the force gradients and dissipation on solvation shells of octamethylcyclotetrasiloxane (OMCTS) molecules on mica.     

Space charge limited current measurements on conjugated polymer films using conductive atomic force microscopy

We describe local (~150 nm resolution), quantitative measurements of charge carrier mobility in conjugated polymer films that are commonly used in thin-film transistors and nanostructured solar cells. We measure space charge limited currents (SCLC) through these films using conductive atomic force microscopy (c-AFM) and in macroscopic diodes. The current densities we measure with c-AFM are substantially higher than those observed in planar devices at the same bias. This leads to an overestimation of carrier mobility by up to 3 orders of magnitude when using the standard Mott-Gurney law to fit the c-AFM data. We reconcile this apparent discrepancy between c-AFM and planar device measurements by accounting for the proper tip-sample geometry using finite element simulations of tip-sample currents. We show that a semiempirical scaling factor based on the ratio of the tip contact area diameter to the sample thickness can be used to correct c-AFM current-voltage curves and thus extract mobilities that are in good agreement with values measured in the conventional planar device geometry.     

Bubble-based acoustic radiation force elasticity imaging

Acoustic radiation force is applied to bubbles generated by laser-induced optical breakdown (LIOB) to study viscoelastic properties of the surrounding medium. In this investigation, femtosecond laser pulses are focused in the volume of gelatin phantoms of different concentrations to form bubbles. A two-element confocal ultrasonic transducer generates acoustic radiation force on individual bubbles while monitoring their displacement within a viscoelastic medium. Tone burst pushes of varying duration have been applied by the outer element at 1.5 MHz. The inner element receives pulse-echo recordings at 7.44 MHz before, during, and after the excitation bursts, and crosscorrelation processing is performed offline to monitor bubble position. Maximum bubble displacements are inversely related to the Young's moduli for different gel phantoms, with a maximum bubble displacement of over 200 microm in a gel phantom with a Young's modulus of 1.7 kPa. Bubble displacements scale with the applied acoustic radiation force and displacements can be normalized to correct for differences in bubble size. Exponential time constants for bubble displacement curves are independent of bubble radius and follow a decreasing trend with the Young's modulus of the surrounding medium. These results demonstrate the potential for bubble-based acoustic radiation force methods to measure tissue viscoelastic properties.     

Contribution to crystallographic slip assessment by means of topographic measurements achieved with atomic force microscopy

In this paper, atomic force microscopy (AFM) is used to quantitatively characterize the plastic glide occurring during tensile deformation of a duplex 2205 stainless steel sample. We demonstrate that an appropriate treatment of the topographic image issued from AFM measurements allows precise and quantitative information about the characteristics of plastic deformation and especially the amount of crystallographic slip.     

Dendrimer-mediated synthesis of platinum nanoparticles: new insights from dialysis and atomic force microscopy measurements

In this work, we use AFM measurements in conjunction with dialysis experiments to study the synthesis mechanism and physical state of dendrimer-stabilized platinum nanoparticles. For characterizing particle size distributions by high resolution transmission electron microscopy and AFM, sample preparation by drop evaporation presumably minimizes the risk of sample bias that might be found in spin coating or dip-and-rinse methods. However, residual synthesis by-products (mainly salts) must be removed from solutions of dendrimer-stabilized metal nanoparticles prior to AFM imaging. Purification by dialysis is effective for this purpose. We discovered, by UV-visible spectrophotometry and atomic absorption (AA) spectroscopy, that dialysis using 'regular' cellulose dialysis tubing (12?000?Da cut-off) used in all previous work leads to substantial losses of poly(amidoamine) (PAMAM) dendrimer (G4OH), PAMAM-Pt(+2) complex, and PAMAM-stabilized Pt nanoparticles. Use of benzoylated dialysis tubing (1200?Da cut-off) shows no losses of G4OH or G4OH-Pt mixtures. We use AFM to see whether selective filtration during dialysis introduces sampling bias in the measurement of particle size distributions. We compare results (UV-visible spectra, AA results, and AFM-based particle size distributions) for a sample of G4OH-Pt(40) divided into two parts, one part dialysed with regular dialysis tubing and the other with benzoylated tubing. Exhaustive dialysis using benzoylated tubing may lead to the loss of colloidal Pt nanoparticles stabilized by adsorbed dendrimer, but not Pt nanoparticles encapsulated by the dendrimer. The comparisons also lead to new insights concerning the underlying synthesis mechanisms for PAMAM-stabilized Pt nanoparticles.     

Local anodic oxidation by atomic force microscopy for nano-Raman strain measurements on silicon-germanium thin films

Nanolithography based on local anodic oxidation (LAO) by atomic force microscopy is a promising technique for patterning strained film nanostructures on the silicon substrates. Due to its versatility and precise control, LAO is suited for preparing well defined calibration structures for local strain measurements. We investigated silicon-germanium patterns prepared by LAO and subsequent selective anisotropic wet etching. By combining the nanolithography and etching, dedicated strain test structures with a line width of 65 nm were achieved and utilized for calibration of tip-enhanced Raman measurements.     

Nanofabrication with atomic force microscopy

Atomic force microscopy (AFM) was developed in 1986. It is an important and versatile surface technique, and is used in many research fields. In this review, we have summarized the methods and applications of AFM, with emphasis on nanofabrication. AFM is capable of visualizing surface properties at high spatial resolution and determining biomolecular interaction as well as fabricating nanostructures. Recently, AFM-based nanotechnologies such as nanomanipulation, force lithography, nanografting, nanooxidation and dip-pen nanolithography were developed rapidly. AFM tip (typical radius ranged from several nanometers to tens of nanometers) is used to modify the sample surface, either physically or chemically, at nanometer scale. Nanopatterns composed of semiconductors, metal, biomolecules, polymers, etc., were constructed with various AFM-based nanotechnologies, thus making AFM a promising technique for nanofabrication. AFM-based nanotechnologies have potential applications in nanoelectronics, bioanalysis, biosensors, actuators and high-density data storage devices.     

Young’s modulus measurements of nanohoneycomb structures by flexural testing in atomic force microscopy

This paper determines the Young’s modulus of nanohoneycomb structures by flexural testing in an atomic force microscope (AFM). Since the cross-sectional area of nanohoneycomb structures varies along the structure, the area moment of inertia is not a constant. The area moment of inertia is also influenced by the porosity of the nanohoneycomb structure. An anodic aluminum oxide (AAO) film is fabricated as a nanohoneycomb structure. Young’s modulus of the AAO film, measured from the results of flexural testing in AFM, is in good agreement with the results of tensile tests in a Nano-UTM (universal testing machine).     

Histogram method for reliable thickness measurements of graphene films using atomic force microscopy(AFM)

Atomic force microscopy(AFM) is a commonly used technique for graphene thickness measurement.However, due to surface roughness caused by graphene itself and variation introduced in AFM measurement, graphene thickness is difficult to be accurately determined by AFM. In this paper, a histogram method was used for reliable measurements of graphene thickness using AFM. The influences of various measurement parameters in AFM analysis were investigated. The experimental results indicate that significant deviation can be introduced using various order of flatten and improperly selected measurement parameters including amplitude setpoint and drive amplitude. At amplitude setpoint of 100 mV and drive amplitude of 100 m V, thickness of 1 layer(1L), 2 layers(2L) and 4 layers(4L) graphene were measured.The height differences for 1L, 2L and 4L were 1.51 ± 0.16 nm, 1.92 ± 0.13 nm and 2.73 ± 0.10 nm, respectively. By comparing these values, thickness of single layer graphene can be accurately determined to be0.41 ± 0.09 nm.     

Lorentz force actuation of a heated atomic force microscope cantilever

We report Lorentz force-induced actuation of a silicon microcantilever having an integrated resistive heater. Oscillating current through the cantilever interacts with the magnetic field around a NdFeB permanent magnet and induces a Lorentz force that deflects the cantilever. The same current induces cantilever heating. With AC currents as low as 0.2 mA, the cantilever can be oscillated as much as 80 nm at resonance with a DC temperature rise of less than 5 °C. By comparison, the AC temperature variation leads to a thermomechanical oscillation that is about 1000 times smaller than the Lorentz deflection at the cantilever resonance. The cantilever position in the nonuniform magnetic field affects the Lorentz force-induced deflection, with the magnetic field parallel to the cantilever having the largest effect on cantilever actuation. We demonstrate how the cantilever actuation can be used for imaging, and for measuring the local material softening temperature by sensing the contact resonance shift.     

Surface roughness, mechanical and tribological properties of ultrathin tetrahedral amorphous carbon coatings from atomic force measurements

Atomic force microscope (AFM), lateral force microscope and AFM-based scratch and wear testing techniques were used to evaluate and compare the surface roughness, tribological and mechanical properties of thin (2.7-43 nm) tetrahedral amorphous carbon coatings prepared by pulsed cathodic arc discharge. It was found that surface roughness of ultrathin (2-8 nm) coatings was mainly determined by the roughness of the Si substrate and their average density strongly depended on their thickness. Poor friction, mechanical properties of thinner (2.7-15 nm) coatings can be associated with their low average density. The dense coatings (>15 nm) had lower friction coefficient, better scratch and wear resistance properties that were independent of their thickness. It appears that the over 15-nm coatings studied are feasible for some wear-resistant and tribological applications.     

Experimental validation of the MODTRAN 5.3 sea surface radiance model using MIRAMER campaign measurements

Radiometric images taken in mid-wave and long-wave infrared bands are used as a basis for validating a sea surface bidirectional reflectance distribution function (BRDF) being implemented into MODTRAN 5 (Berk et al. [Proc. SPIE5806, 662 (2005)]). The images were obtained during the MIRAMER campaign that took place in May 2008 in the Mediterranean Sea near Toulon, France. When atmosphere radiances are matched at the horizon to remove possible calibration offsets, the implementation of the BRDF in MODTRAN produces good sea surface radiance agreement, usually within 2% and at worst 4% from off-glint azimuthally averaged measurements. Simulations also compare quite favorably to glint measurements. The observed sea radiance deviations between model and measurements are not systematic, and are well within expected experimental uncertainties. This is largely attributed to proper radiative coupling between the surface and the atmosphere implemented using the DISORT multiple scattering algorithm.