Bacterial adhesion to a surface is a critical step in the formation of a biofilm. Biofilms are a protective community for microorganisms and they are of concern in a variety of fields, from biomaterial-associated infection to applications in the food and environmental industries. We used atomic force microscopy (AFM) to measure the forces of adhesion of Pseudomonas fluorescens Pf0-1 with a silicon nitride tip, and to characterize the properties of surface polymers of P. fluorescens. Specifically, we investigated the role of LapA, a putative outer membrane-associated adhesin that is necessary for biofilm formation, on bacterial adhesion and biopolymer properties. P. fluorescens strains with various LapA levels on the cell surface, as well as different LapA variants, were used in these studies. The wild-type strain had a median force of adhesion of 0.60 nN, while the lapA mutant showed significantly decreased adhesion forces of 0.28 nN, highlighting the role of the LapA protein in cellular adhesion. We looked at the role of the calcium binding domain on adhesion, and found that a LapA variant lacking the Calxβ domain, showed an adhesive force similar to the wild type. This indicates that removal of the calcium-binding domain did not impair the ability of this strain to have strong adhesion forces, and furthermore, this variant was unaffected for attachment to a abiotic surface. A strain deleted for the LapG protease, which is unable to release LapA and therefore has increased levels of the LapA protein on the cell surface, exhibited the highest adhesion forces with the model AFM tip, namely 1.32 nN. Introducing a protease binding site (TEV) to the LapG mutant slightly decreased the adhesion of the strain. Treatment of the strain expressing the TEV site-containing mutant of LapA with AcTEV protease resulted in reduced adhesion forces compared to the untreated strain, suggesting that removal of this portion of the LapA protein decreased measured adhesion forces. These experiments provide new insight into the role of LapA protein on bacterial adhesion, and will ultimately lead to a better understanding of biofilm formation.

There are a number of technologies that couple nanomaterials with biological molecules. For example, the formation of stable dispersions of single-walled carbon nanotubes (SWCNTs) and single-stranded DNA (ssDNA) makes SWCNTs highly compatible for in vivo systems in biomedical applications and also provides a means for tube sorting and positioning on surfaces. It is important to quantify the interactions between these two components in order employ these hybrids efficiently and safely. We will describe single molecule force spectroscopy experiments that directly measure the peeling forces of individual synthetic single-stranded DNA oligomers from graphite - a two-dimensional analogue of the surface of carbon nanotubes. Considering these peeling forces in the context of an equilibrium model for peeling a single freely jointed polymer chain from a frictionless substrate, we determined a ranking of the effective average binding energy per nucleotide for all four bases as Tge;A>Gasymp;C (11.3 ± 0.8 k_BT, 9.9 ± 0.4 k_BT, 7.9 ± 0.2 k_BT, and 7.5 ± 0.8 k_BT, respectively). Furthermore, we used Brownian Dynamics simulations of a freely jointed chain to study the peeling of a polymer from a frictionless surface under both force and displacement control. At sufficiently slow peeling rates (<15 mu;N/s under force control and <1.4 mm/s under displacement control), these simulations confirm the assumptions underlying the equilibrium model. The model is further used to study deviations from equilibrium and the effect of AFM probe fluctuations, in particular, how these factors influence our ability to detect distinct sequences by force spectroscopy.

Live cells and many biological samples readily deform under the minimum force required to perform an AFM measurement. To overcome this problem we developed a new type of cantilever with exceptionally low force noise in liquid, enabling gentle imaging at high temporal and spatial resolution. Our cantilevers are encased in a micro-fabricated transparent Silicon Nitride layer. Surface tension prevents liquid from entering into the encasement, thus keeps the resonator dry and only few micrometers of the tip apex protrude from the encasement to interact with the sample. This maintains high resonance frequency and quality factors as operating in air. Compared to the performance of regular cantilevers the force noise is decreased by more than one order of magnitude, reaching minimal detectable forces of 12 fN/radic;Hz in liquids. Such low noise expands the frontier of measurements in solution enabling high-speed imaging of soft material surfaces and ultrahigh precision force spectroscopy. As the length of the free cantilever is controlled by the release process of a sacrificial layer, ultra short cantilevers with resonance frequencies >1 MHz as well as long and soft cantilevers (<20kHz, 0.03N/m) can be realized. The former are suitable for gentle high-speed imaging and the high force sensitivity reduces the risk of deforming or destroying soft matter during the measurement. The later soft cantilevers are ideal candidates for ultra sensitive force spectroscopy to study the energy landscapes in folding and unfolding of proteins or binding forces in ligand-receptor complexes. Since the encasement is fabricated using a transparent silicon nitride layer and the cantilever length and width are on the order of 10 microns, the cantilever position can be detected in any conventional AFM setup using optical beam deflection.

Atomic force microscopy (AFM) is developing as a family of complimentary operation modes based on the detection of cantilever deflection or its vibrational parameters (amplitude, frequency) caused by tip-sample interactions. The deflection, amplitude or frequency at a set-point level can be applied in feedback control for high-resolution profiling of surfaces, visualization of surface structures and compositional mapping of heterogeneous samples. The probe deflection is directly related to the interaction force, and it can be detected not only in the quasi-static contact mode but also in the oscillation modes operating at non-resonant and resonant probe frequencies. These modes have been successfully applied for studies of different materials, yet they have some limitations that will be briefly discussed. The amplitude and frequency modulation modes are also broadly used for studies in air and under liquid. High-sensitivity of amplitude, phase and frequency of the resonant probe oscillation to tip-sample forces and force gradient are utilized in these applications. In the optimization of AFM experiments, the practitioners are often challenged in choosing the appropriate mode. In an attempt to facilitate their choice, we have performed a theoretical analysis of the oscillation modes in the framework of the Euler-Bernoulli description of the interacting probe assisted by asymptotic Krylov-Bogoliubov-Mitropolsky approach [1]. This analysis allows classification of the AFM mode and finding the relationship between the amplitude, phase and frequency of the probe, the level of force interactions and mechanical sample properties. These results are paralleled with the experimental imaging of soft materials (biological specimen, polymers) and rigid samples in the non-resonant and resonant oscillation modes. A particular emphasis is made on the comparative ability of these modes in visualization of soft and fragile top layers. The problems of elucidating quantitative mechanical data (elastic modulus and work of adhesion) from AFM experiments will be also discussed. 1. Belikov, S., Magonov S. “Classification of Dynamic Atomic Force Microscopy Control Modes Based on Asymptotic Nonlinear Mechanics” Proceedings American Control Society, St. Louis, 979-985, 2009.

Instrumental drift in atomic force microscopy (AFM) remains a critical issue that limits the precision and duration of experiments. Previously, we have used an active optical stabilization technique to improve positional stability by a factor of 100. However, force drift also occurs via uncontrolled deflection of the zero-force position of the cantilever over time. We found that the primary source of force drift for a popular class of soft cantilevers is their gold coating, even though they are coated on both sides to minimize drift. When the gold coating was removed with a simple chemical etch, the force drift after two hours was reduced from 900 nm for gold-coated cantilevers to 70 nm (N =10; rms) for uncoated cantilevers. Although removing the gold led to ~10-fold reduction in reflected light, short-term (0.1-10 s) force precision improved. Moreover, improved force precision did not require extended settling; most of the cantilevers tested achieved sub-pN force precision over a broad bandwidth (0.01-10 Hz) just 30 minutes after loading. We demonstrated the usefulness of the lowered drift with force curves on DNA and other biological macromolecules. Thus, by simply using uncoated cantilevers, high-precision AFM measurements can be made after minimal settling time.

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 on mechanically unfold individual proteins by pulling from two points. However, its correlation with protein structure requires additional measurements. Here, we apply a force-distance-curve -based AFM imaging mode to simultaneously acquire structural and elasticity information of membrane proteins of bacteriorhodopsin (1) and AQP0 and gap junction connexons in native eye lens membranes (2). Furthermore, using dynamic force spectroscopy AFM with a peptide (NTVD) that mimics loop E2 of connexons covalently linked to the AFM tip, we characterize the binding strength of the gap junction interaction. Force curves at various retraction speeds were acquired to determine the dissociation kinetics of the peptide-connexon interaction, while adhesion probability measurements at different contact times revealed the binding kinetics. The relatively fast intrinsic dissociation rate (koff) inferred a rather dynamic inter-connexon interaction, while the slow association rate (kon) probably reflects the restricted mobility and degrees of freedom of the connexons in the densely packed organization observed in native gap junction plaques and the reduced flexibility and dimensions of the extracellular loops. Our results suggest that gap junction formation may occur before plaque formation. (1) Felix Rico, Chanmin Su & Simon Scheuring. Mechanical mapping of single proteins at the submolecular level, Nano Letters, 2011, 11 (9): 3983-3986. (2) Felix Rico, Adai Colom, Laura Picas & Simon Scheuring. In preparation (3) Felix Rico, Atsunori Oshima, Peter Hinterdorfer, Yoshinori Fujiyoshi & Simon Scheuring. Two-dimensional kinetics of inter-connexin interactions from single molecule force spectroscopy, J Mol Biol, 2011, 412 (1): 72-79

Ganglioside 1 (GM1) is an essential component of eukaryotic plasma membrane that can organize into micro or nanodomains. This lipid has been previously shown to partition in lipid-ordered phase using AFM [1,2]. Recently, we have used high-speed atomic force microscopy [3] to revisit both partition and diffusion of GM1 nanodomains within supported lipid bilayer. We used GM1 incorporated into a lipid bilayer composed of a mixture of DOPC/DPPC (1:1) forming lipid phase separation. Under these conditions, we observed small domains of GM1 diffusing in the DPPC phase and that we characterized in terms of size and diffusion. References [1] Vié, V., Van Mau, N., Lesniewska, E., Goudonnet, J. P., Heitz, F., and Le Grimellec, C. (1998) Langmuir 14, 4574-83 [2] Milhiet, P.E., Vié, V., Giocondi, M.C. and Le Grimellec, C. (2001). Single Molecule 2, 109-112. [3] Ando T., Uchihashi T., and Fukuma, T., Prog. Surf. Sci. 83(7-9) 337-437 (2008).

Atomic force microscopy is widely used to study lipid membrane and monolayers and their interactions with proteins. Kelvin Probe Force Microscopy (KPFM) specifically addresses electrostatic properties of materials and has a great potential to bring new discoveries in biomedical research, but currently has limited biological applications. In this work, we report one of a few applications of Kelvin probe force microscopy (KPFM) to study complex structures of lipid films and lipid-protein interactions. Molecular arrangement of lipids and proteins gives rise to complex film morphology as well as distinct electrical surface potentials, which may rule many biological processes and diseases. Using AFM and Frequency Modulated - KPFM (FM-KPFM) we showed that cholesterol creates nanoscale electrostatic domains which induce preferential binding of amyloid peptide 1-42 and may be important to understand the mechanism of amyloid toxicity in relation to Alzheimer&’s disease. Earlier we observed similar electrostatic domains induced by cholesterol, which impaired the function of pulmonary surfactant (Langmuir 2010). Here we show that this electrostatic effect of cholesterol is not specific to only pulmonary surfactant films, but is also present in model lipid systems and plays an important role in amyloid-lipid interactions. We postulate that this previously unknown nanoscale electrostatic effect of cholesterol is a fundamental property, which, in turn, may greatly influence the interactions of lipid membranes with other charged molecules and nanoparticles. 1. B.Moores, F.Hane, L.M.Eng, Z.Leonenko, Kelvin probe force microscopy in application to biomolecular films: Frequency modulation, amplitude modulation, and lift mode, Ultramicroscopy, 2010, 110(6), 708-711. 2. E.Finot, Y.Leonenko, B.Moores, L.M.Eng, M.Amrein, Z.Leonenko. Effect of cholesterol on electrostatics in lipid-protein films of a lung surfactant, Langmuir, 2010, 26 (3), 1929-1935. 3. E.Drolle, R.Gaikwad, Z.Leonenko, Nanoscale electrostatic domains in cholesterol-laden lipid membrane create a target for amyloid binding, Biophys. J. Letters, 2012, accepted.

Trinuclear gold(I) pyrazolates can bind strongly to electron-poor aromatic compounds. Having such complexes immobilized on graphene or a carbon nanotube network can therefore be a way to build selective and sensitive sensors. Such sensors could be used for the detection of chlorinated aromatic compounds, of interest for environmental sensing, and nitroaromatic compounds, of interest for transportation security. Trinuclear gold(I) complexes were functionalized with alkyl chains to enable their self-assembly on a Highly Oriented Pyrolitic Graphite (HOPG) surface. This surface can be seen as a model system for graphene and carbon nanotubes. A molecular layer was formed of the trimeric gold(I) complexes at the 1-octanoic acid/HOPG interface, and, to better understand the assembly process, these layers were subsequently studied on the molecular level by Scanning Tunneling Microscopy (STM). The STM studies show that the trinuclear gold(I) complexes can be present in differerent morphologies and give valuable insights in the dynamic processes that occur during the formation and after the layer is formed. Obtaining details of such a process on the molecular level would be very hard, if not impossible, by other techniques. Such processes and the exact morphology of the molecular layer can be expected to have a significant effect on the implementation of such layers in a sensor geometry, illustrating the importance of studying these processes on the single molecule level.

We report quantitative infrared spectroscopy on polymer nanostructures as small as 15 nm using atomic force microscope infrared spectroscopy (AFM-IR). In AFM-IR, an atomic force microscope tip detects the rapid thermomechanical expansion of an absorbing material irradiated by a short IR laser pulse. This rapid expansion shocks the cantilever into oscillation, the magnitude of which corresponds to the sample spectral absorbance. As the size of the absorbing feature decreases, the cantilever oscillation magnitude decreases, making it difficult to measure infrared absorption of structures less than 100 nm tall. Using numerical simulations of the polymer thermomechanical response and the cantilever mechanical response, we found that the smaller polymer features cool more quickly following the 10 ns laser pulse, and that the shorter cooling times lead to larger excitation of higher cantilever flexural modes. Polymer structures with sizes between 0.1 - 1 um cooled within 0.1 - 10 us. To test these predictions, we fabricated polyethylene (PE) nanostructures of size 100 nm, 600 nm, and 2000 nm and measured their properties with AFM-IR. The measurements confirmed the link between polymer nanostructure size, thermomechanical response, and the flexural modes excited. Our key insight is that the signal to noise of the measurements on smallest nanostructures can be increased by capturing cantilever dynamics localized to higher harmonics within a short time duration just after the laser pulse. When a 100 nm PE nanostructure is interrogated with AFM-IR at 2920 cm-1, the cantilever sensitivity can be increased by a factor of 6 by including the response of the first 8 cantilever flexural modes. To further improve the cantilever signal to noise, we use a continuous Morlet wavelet transform to window the cantilever response in both the time and frequency domain. With this approach, the signal to noise can be improved by a factor of 10. Finally, we use these advances to demonstrate quantitative IR characterization of a PE nanostructure of size 15 nm in the spectral range 2700 - 3200 cm-1.

Since its invention in 1982, the STM has relied upon piezoelectric elements to move the scanning tip and track the topography of a surface with a resolution of a few pm. However, a typical STM image is not a quantitatively accurate map of the local surface. For example, straight features will often appear curved, even where drift should be minimal. Likewise, the forward and reverse scans of the same area do not typically overlap, which has been ascribed to hysteresis. While a distorted image provides qualitative information which is extremely useful for scientific purposes, for any metrological application where accurate positioning of the tip is important, such as measurement of structure dimensions, local spectroscopy of a single surface feature, or STM-driven patterning with atomic precision, these distortions pose a significant source of error. In a piezoelectric material, an applied voltage causes the crystal to change its length with a linear response. However, the length change does not occur instantaneously after the voltage is applied; the final ca. 10% occurs slowly over the course of many seconds. This phenomenon, known as piezoelectric creep, is the source of many image distortions, and we are developing methods for its measurement and correction. We have a homemade STM, based on the Lyding design, with a piezoelectric tube scanner, and use H-terminated Si(001) for the sample. To measure creep on the timescale of seconds, the tip is first allowed to settle to remove any accumulated position error. Then the tip moves abruptly sideways a certain distance (40-200 nm), and immediately starts scanning an image. The top of the image will show strong curvature in the dimer rows, resulting from creep. This curvature is fitted to exponential decays, giving time constants and amplitudes as a function of the initial displacement. For creep much faster than a single linescan (0.2 s), we compare the distortions in the forward and reverse scan directions during scanning. By analysing the offsets in positions of surface features such as dimer row peaks and defects, we are able to fit this fast creep to another exponential decay. With known creep parameters, we can apply a real-time piezo voltage correction, which decays over time to reflect the known creep decay constants. In this way, we generate images which are a more accurate map of the local surface, without curvature at the top of the image, and with the forward and reverse scans overlapping. Creep-corrected STM will be a major step forward towards allowing quantitative measurements of nanostructure dimensions, and accurate tip positioning over any chosen atom.

Reduction of metal oxides is a reaction of removing lattice oxygen and plays a critical role in many technological processes including heterogeneous catalysis, metallurgy, and electronic device fabrication. However, the microscopic processes leading to the reduction of metal oxides are still significantly unclear. By employing a combination of in-situ scanning tunneling microscopy (STM) and x-ray photoemission spectroscopy (XPS), we investigate the atomistic processes governing the reduction of oxygen chemisorbed phases on Cu(110). By varying the oxygen partial pressure and oxidation temperature, the Cu(110) surface can be oxidized to form either (2x1)-O or (6x2)-O phases or their co-existence. Following the oxidation, hydrogen is introduced to reduce the oxygen chemisorbed phases. The surface structure changes are monitored by STM imaging while the oxygen desorption kinetics are measured by XPS under the reduction conditions. The effect of hydrogen gas pressure and the reduction temperature on the reduction kinetics and surface structure evolution is examined in detail. The experimental observations of the relative stability and reduction kinetics of the two oxygenated surface phases under the reduction reactions are correlated with the theoretical modeling of the density-functional theoretical calculations and kinetic monte carlo simulations.

The surface potential is a key electrical state variable that affects energy of surface- or interface-confined charges and thus the energetics of charge transport. It also provides a direct link between microstructural features and electrical properties. Therefore, probing the surface potential variation and correlating it with microstructure in device-active semiconductor thin films offers a unique approach to understanding fundamental structure-property correlations. In this work, we have employed Kelvin Probe Force Microscopy (KFM) and Electrostatic Force Microscopy (EFM) to study the surface potential in ultrathin films of a benchmark organic semiconductor pentacene. Both KFM and EFM allow spatial mapping and quantification of surface potentials as well as correlation with simultaneously acquired topographic images that reveal microstructure. The inter- and intra-layer surface potential variations in pentacene films thermally deposited on different dielectric substrates have been mapped and quantified. The influence of deposition conditions, i.e., the substrate temperature, on the surface potential has been investigated. We have found that the surface potential of pentacene films strongly depends on the substrate type and deposition conditions. Furthermore, the surface potential variations of the films have been correlated to the microstructure, in particular, the homoepitaxial relationship between the second and first pentacene monolayers. We demonstrate that there is a general one-to-one correlation between homoepitaxy and surface potential in pentacene films deposited on a variety of substrates, which has important implications for other polycrystalline organic semiconductor films. The significance of our work is that it reveals a direct connection between microstructure and a key electrical property, i.e., the surface potential, and thus provides a framework for understanding how microstructure can impact carrier transport in organic semiconductors.

A substantial contribution to the device current in organic solar cells comes from regions in the vicinity of the electrode, typically tens to hundreds of microns from the electrode edge[1]. We discuss the role of this peripheral photocurrent in the context of probing the bulk heterojunction utilizing a combination of atomic force microscopy, near field optical microscopy, near field photocurrent microscopy. A quantitative analysis of the heterojunction geometry using angular Fourier transformation techniques has been developed to rationalize optimum blend concentration in crystalline and amorphous donor systems [2-3]. We also discuss device geometries that utilize the peripheral current in patterned devices fabricated through melt based electrode deposition techniques. References: [1] D. Gupta, et al., "Area dependent efficiency of organic solar cells," Applied Physics Letters, vol. 93, Oct 20 2008. [2] S. Mukhopadhyay and K. S. Narayan, "Rationalization of donor-acceptor ratio in bulk heterojunction solar cells using lateral photocurrent studies," Applied Physics Letters, vol. 100, p. 163302, 2012. [3] S. Mukhopadhyay, et al., "Direct Observation of Charge Generating Regions and Transport Pathways in Bulk Heterojunction Solar Cells with Asymmetric Electrodes Using near Field Photocurrent Microscopy," The Journal of Physical Chemistry C, vol. 115, pp. 17184-17189, 2011/09/01 2011.

The field of spintronics focuses on using both spin and charge to transmit information and has a number of applications particularly in magnetic memory devices. In such devices, it is important to develop 1D structures which could play the role of wires transmitting the information via spin rather than current. This setup could provide a new route in current free memory devices. This work realizes one method of spin transport via mixed Co/Fe 1D chains on Ir(001). Pure Co and Fe along with coupled Co-Fe chains were examined via spin-polarized scanning tunneling microscopy (SPSTM) and spectroscopy. Both Fe and Co self-assemble into bi-atomic chains on the Ir(001)-(5x1) reconstructed surface. The structure of the Ir(001)-(5x1) allows for three possible 1D structures in the trenches of the reconstruction and the Fe grows as an Inner Hollow (IH) site chain while Co exhibits some IH site structures but is predominately in the Zig-Zag (ZZ) stacking which has broken symmetry along the chain axis. Thus, when co-deposited the materials can often be distinguished in topography and always be identified by spectroscopy due to differences in the local density of states. The Co exhibits a ferromagnetic ground state that is stable at 8K. First-principles calculations of these chains show an intriguing interplay between the chain structure and easy magnetization direction. The magnetic structure of the Fe chain is more complicated and spin-resolved measurements show a periodic spin-spiral along the entire length of the chain, which is stabilized in an applied field but fluctuates at a speed greater than the time resolution of the STM in zero applied field [1]. When both Fe and Co are co-deposited on the surface, the Co stabilizes the Fe spin-spiral and information about the magnetic state of the Co can be transmitted via the Fe spin-spiral. [1] M. Menzel et al. Phys. Rev. Lett. 108, 197204 (2012).

In this work, the austenite-martensite phase transformation in 316 stainless steel (SS) was studied in situ at room temperature in a scanning probe microscope (SPM) during application of uniaxial tensile stress. Austenitic steel is known to be metastable to the martensitic phase transformation at room temperature. Specially designed SS dogbones were fabricated to allow imaging of structural and phase transformations while tensile straining the sample using a small commercial tensile stage. The surface was polished to a mirror finish and etched to allow identification of individual grains. Since martensitic steel is a ferromagnetic, magnetic force microscopy (MFM) was use to identify the distribution of the martensitic phase as a function of strain. A dogbone was strained-to-break (over 20 percent strain) ex situ in order to determine the maximum strain allowable, to avoid possible damage to the SPM tip or scanner. A fiduciary indent in the surface was used to maintain imaging location. Images and tensile stress-strain data was collected up to a strain of 18%. The dramatic increase in magnetic structure and three-dimensional deformation of the surface grains will be presented.

The local mechanical properties of ferritic and austenitic domains in a duplex stainless steel are locally studied by nanoindentation. The elastic and plastic properties of the two phases are determined. Without any special surface treatment (chemical or electrochemical), the austenitic and ferritic domains present in the duplex stainless steel are distinguished using magnetic force microscopy. The magnetic scans allow nanoindentation results to be assigned to the respective phases, yielding the local mechanical properties of the duplex steel. The magnetic scans also show a sharp transition between the phases that is maintained even inside indentations. The ferrite phase is found to supersede austenite in the elastic modulus, hardness, and strain-hardening exponent, while both phases possess similar yield strength. Interface properties are a weighted average of the phase properties.

Current developments of the microelectronics industry require controlling the morphology of materials at the nanoscale. This is the case for ferroelectric materials in which several one-dimensional ferroelectric structures (nanorods, nanowires, nanotubes, and self-assembly of zero-dimensional nanoislands) have been studied. Within this context, we have been exploiting the role of nano- and mesoporosity in ferroelectrics. Preliminary results clearly revealed that nanoporosity influences the structure and physical properties of BaTiO3. [1] Besides that, ordered meso- or nanordered porous thin film arrays may be used as ferroelectric platforms for multifunctional purposes. Due to exceptional spatial resolution, Scanning Probe Microscopy (SPM) techniques and in particular Piezoresponse Force Microscopy (PFM) have become an important high resolution characterization tool for visualization of domain structures in ferroelectric thin films at the nanoscale. In this work, Vertical Piezoresponse Force Microscopy (VPFM), Lateral Piezoresponse Force Microscopy (LPFM), and Switching Spectroscopy Piezoresponse Force Microscopy (SSPFM) are used to characterize the piezoelectric and ferroelectric properties of mesostructured PbTiO3 thin films prepared by sol-gel and evaporation-induced self-assembly methodologies. [2] The thin films were thermally treated at different temperatures in order to assess the effect of the microstructure crystallization evolution on the ferroelectric properties. PFM results clearly demonstrate the existence of two distinct areas, which exhibit different local piezoelectric properties. The area with strong out-of-plane piezoresponse increases with the thermal treatment temperature, as well as the tetragonal phase. The ferroelectric behavior in these samples was also demonstrated by the typical square-shaped piezoelectric hysteresis loops. Some of these hysteresis loops exhibit imprint and the relation between this vertical and lateral shift is related to the processing conditions and porosity. These mesoporous thin films were compared with dense PbTiO3 thin films prepared under the same conditions. PFM analysis was complemented with structural and microstructural studies by X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy, and Raman Spectroscopy. The role of mesoporosity in the ferroelectric behaviour of PbTiO3 is discussed. Due to its mesostructure and strong piezoelectric and ferroelectric behaviour, these thin mesoporous films can be used as platforms to obtain a new generation of multifunctional ordered composite materials with an original architecture. [1] Hou et al, Chem. Mater., 2009, 21, 3536-3541. [2] P. Ferreira, R. Z. Hou, A. Wu, M.-G. Willinger, P. M. Vilarinho, J. Mosa, C. Laberty-Robert, C. Boissière, D. Grosso, C. Sanchez, Langmuir, 2012, 28, 2944.

Scanning nonlinear dielectric microscopy is a powerful technique for imaging polarization distribution on surface of ferroelectrics, accumulated charges (memorized electrons and holes) in flash memories and dopant distributions in semiconductor devices with sub-nanometer resolution. The high resolution imaging is enabled by measuring the nonlinear dielectric constants under a sharp metal tip attached to a GHz-range LC oscillator. The oscillation frequency of the probe is modulated by the tip-sample capacitance variation, which has nonlinear dependence on the externally applied ac electric field. The lateral resolution of SNDM gets higher and SNDM senses shallower area by measuring the higher harmonics. This is understood as the follows. The amplitude of n-th harmonic (nomega;p) is proportional to the n-th power of electric field. The spatial distribution of n-th power of electric field under the tip is more concentrated, with increase of the harmonic number n. As a result, the resolution becomes higher through the measurement of super-higher harmonics. In order to make the resolution much higher, we tried to measure up to 4omega;p signal. However, the signal becomes weaker when we measure the higher harmonics. Therefore, in order to measure the 4omega;p signal, we have to develop more sensitive probe. The sensitivity of probe (frequency deviation from the center frequency of SNDM probe oscillator) is proportional to the oscillating center frequency. Thus we developed the new probe whose center frequency is higher than the conventional one. The oscillating center frequency of the new probe is about 4 GHz. By using this new probe, we present the new experimental results by measuring up to 4omega;p signal. We observed a congruent LiTaO3 (CLT) multi-domain single crystal, which is one of ferroelectric material in air by measuring from 1omega;p to 4omega;p. By comparing images, it is understood that, with the increase of the amplitude of applied voltage from 7Vp-p to 11 Vp-p, very strong nonlinear dielectric effect arises and the clearer domain structures with high contrast were observed even at the super higher harmonic (3omega;p and 4omega;p). Next, comparing the images from 1omega;p to 4omega;p, we confirmed that the thickness of domain boundaries were observed more thinly in higher order image than lower order one, regardless the amplitude of applied ac voltages. The minimum width among the many observed domain boundaries was about 10nm in 1omega;p image and 5nm in 4omega;p image. This means the lateral resolution of SNDM is improved by measuring the super-higher harmonics. Finally, we also observed the cross-section of n-channel MOSFET with the channel width of 80 nm. In higher order measurement, the dopant distribution was observed more clearly than lower order measurement. And very fine structure was resolved in super higher-order imaging. Therefore, super higher-order measurement is very useful for analysis of the ferroelectric materials and semiconductor devices.

Broad implementation of SOFCs systems requires lowering of operational costs and increase of life times, necessitating search for new materials and device solutions. Achieving this goal, in turn, requires understanding microscopic and atomistic mechanisms of fuel cell operation as a necessary step in knowledge-driven design and optimization of SOFC materials and architectures. Recently, a scanning probe microscopy approach for probing local electrochemical functionality was suggested and applied for SOFC cathode and electrolyte materials. In electrochemical strain microscopy (ESM), measured is electrochemical strain induced by a biased conductive tip in contact with the surface. detection Measurements of ESM signal as a function of time and bias gives rise to a broad set of time- and voltage spectroscopies that allow the spatial variability of local electrochemical processes to be mapped. In this work, the temperature and bias evolution of ESM signal on a solid electrolyte film has been studied. The electrochemical hysteretic loops show increase in area with temperature which imply an increase in the surface ionic activity. At higher measurement temperatures, we observe evidence of increased relaxation which should be related to higher rates of ionic diffusivity. The shape of the hysteretic loops is a result of direct interplay between these two competing effects. Understanding the structure-functionality correlation in fuel cell materials requires probing all aspects of temperature and bias dependent electronic and ionic transport and electrochemical reactivity over broad voltage and temperature ranges on the length scales of individual extended defects, and effectively constructing a phase diagram for a highly localized volume of material. In this work, we suggest a new paradigm for high veracity, high throughput probing of these phenomena on the sub-10 nm level. This material is based upon work supported by research division of Materials Science and Engineering, Office of basic energy sciences, DOE.

We provide experimental evidence of the excitation of sub-harmonics in ambient conditions amplitude modulation atomic force microscopy (AM AFM) [1-3]. An understanding of sub-harmonic excitation could lead to experimental determination of the presence of water on the sample, chemical affinity and mechanical properties of materials with the high spatial resolution characteristic of AM AFM. Several experimental regimes of interaction, i.e. non-contact mode, attractive force regime and repulsive force regime, are shown to excite sub-harmonics and display a distinct signature that could be related to properties in the long and short range, i.e. physical/chemical and environmental properties [4]. From the experimental data, we decouple contributions related to relative humidity, i.e. capillary interactions[2], chemistry, i.e. SiO2- SiO2, Al-Al, Au-Au, physical, i.e. non-contact and attractive regime, and chemical/mechanical, i.e. repulsive regime where mechanical contact occurs. [1] Chiesa M, Gadelrab K, Verdaguer A, Segura J J, Barcons V, Thomson H N, Phillips M A, Stefancich M and Santos S 2012 Energy dissipation due to sub-harmonic excitation in dynamic atomic force microscopy. [2] Santos S, Barcons V, Verdaguer A and Chiesa M 2011 Sub-harmonic excitation in AM AFM in the presence of adsorbed water layers Journal of Applied Physics 110 114902-11 [3] Barcons V, Verdaguer A, Font J, Chiesa M and Santos S 2012 Nanoscale capillary interactions in dynamic atomic force microscopy Journal of Physical Chemistry C 16 7757-66 [4] Chiesa M, Gadelrab K, Stefancich M, Armstrong P, Li G, Souier T, Thomson N H, Barcons V, Font J, Verdaguer A, Phillips M A and Santos S 2012 Investigation of Nanoscale Interactions by Means of Sub-harmonic Excitation.

Nanocomposites play an increasing role as structural materials; especially the use of high-strength nanofillers such as carbon nanotubes or graphene in polymers show great promise to make novel high-performance lightweight materials. However, a rigorous understanding and analysis of these materials is difficult due to the lack of an experimental technique with good enough sensitivity and resolution to detect individual of these nanoscale fillers inside the polymer matrix as the material is under load. Without such information, material development has to focus on “trial and error” optimization, trying to improve macroscopic material properties. Carbonaceous nanoparticles embedded in a polymer matrix do usually not provide very strong contrast in electron micrographs. However, since they exhibit very stark differences in mechanical properties, such as stiffness, we advocate force microscopy, essentially a mechanical technique, as a well-suited tool to investigate these systems. Using force modulation microscopy, we can reveal single-layer graphene oxide (GO) sheets with diameters <100 nm with good contrast when they are embedded at the surface of a polymer. We included a mechanical straining stage into our commercial AFM, which allows us to study the strain response of graphene-based nanocomposites at the level of individual sheets. By monitoring sheet positions, the local matrix strain can be measured for different externally applied strains. Moreover, our resolution is good enough to visualize the degree of strain in every single GO sheet, which allows us to determine the effectiveness of the load transfer across the graphene/polymer interface quantitatively. We envision that our approach will provide means to predict macroscopic material properties on the basis of nanoscale behavior, and that it will be used to test predictions of nanocomposite models not only at the macroscale, but also at the nanoscale, which is important to verify multi-scale models.

Energy analysis of the cantilever dynamics in atomic force microscopy is a powerful approach to study energy dissipative modes at the nanoscale. While viscosity and surface energy hysteresis were proposed to be the two main mechanisms of energy dissipation during mechanical contact [1,2], getting quantifiable results that are material dependent is still challenging [3-5]. In this study, we provide a framework to detect the presence of these two modes of energy dissipation by studying the total amount of energy dissipated and the phase lag of the cantilever&’s response [4]. If these processes are found to prevail in the contact, we show that sample deformation can be quantified with, in principle, picometer resolution [6]. The method is based on a formalism derived from the energy conservation principle and further allows extracting the dissipative coefficients, i.e. the viscosity of the sample and the variation in surface energy during tip approach and tip-retraction. The method is validated with the use of numerical simulations. Experimentally, we have extracted the sample deformation, viscosity and variation is surface for a variety of samples: steel, SiO2, graphite, mica and polypropylene. Robust quantification of fundamental dissipative parameters with nanoscale spatial resolution could lead to the development of theoretical models at the atomic-continuum interface. [1] Garcia R, Goacute;mez C J, Martinez N F, Patil S, Dietz C and Magerle R 2006 Identification of Nanoscale Dissipation Processes by Dynamic Atomic Force Microscopy Physical Review Letters 97 016103-4 [2] Garcia R, Magerele R and Perez R 2007 Nanoscale compositional mapping with gentle forces Nature Materials 6 405-11 [3] J.Gomez C and Garcia R 2010 Determination and simulation of nanoscale energy dissipation processes in amplitude modulation AFM Ultramicroscopy 110 626-33 [4] Gadelrab K R, Santos S, Souier T and Chiesa M 2012 Disentangling viscous and hysteretic components in dynamic nanoscale interactions Journal of Physics D 45 012002-6 [5] Santos S, Gadelrab K R, Souier T, Stefancich M and Chiesa M 2012 Quantifying dissipative contributions in nanoscale interactions Nanoscale 4 792-800 [6] Santos S, Gadelrab K, Barcons V, Stefancich M and Chiesa M 2012 Quantification of dissipation and deformation in ambient atomic force microscopy New Journal of Physics Under Review

Scattering near-field scanning optical microscopy called ANSOM or sSNOM has been applied to look at plasmonic distribution. Unfortunately, the probes that need to be used in order to effectively scatter the plasmonic signal have significant perturbation on the plasmonic propagation because of the need to use probes with high dielectric constant to obtain effective signal to noise in such scattering experiments. In this paper, we will demonstrate the application of our development of multiprobe scan probe microscope technology for effective localized illumination of plasmonic structure with an apertured NSOM probe which produces all k-vectors. The propagating plasmons are imaged with an active fluorescent material embedded in a glass probe [1] with minimal perturbation of the plasmonic propagation. The results indicate that localized apertured NSOM illumination and active apertureless monitoring of plasmons has significant potential for investigating plasmonic structures. [1] A. Lewis and K. Lieberman, "Near-field Optical Imaging with a Non-evanescently Excited High-brightness Light Source of Sub-wavelength Dimensions," Nature 354, 214 (1991).

Advanced polymer-based materials require improved atomic force microscopy (AFM) methods to characterize their temperature- and frequency-dependent properties at the nanoscale. Here, we perform contact resonance force microscopy (CR-FM) measurements with heated tip AFM (HT-AFM) cantilevers in order to obtain rapid, temperature-dependent mechanical data. Nanoscale thermal analysis with HT-AFM uses specialized cantilevers to achieve local heating, while quantitative nanomechanics with CR-FM measures the frequencies of the cantilever resonance in contact. To combine methods, we investigate issues such as how the geometry and spring constant of commercial HT-AFM cantilevers affect CR-FM methods. We perform dynamic finite element modeling of HT-AFM cantilevers with accurate dimensions for a range of tip-sample contact stiffnesses. The results enable flexural and torsional contact resonances to be correctly identified and allow the vertical and lateral contact stiffnesses to be more accurately determined. Also, CR-FM frequency data are obtained with commercial HT-AFM cantilevers and analyzed with a closed-form expression for Euler-Bernoulli beam dynamics. These experimental results are compared to the finite element predictions to determine the applicability of existing CR-FM models to HT-AFM cantilevers. Results also provide insight into measurement challenges such as operating in a regime of high lateral contact stiffness and accommodating large changes in contact area during temperature sweeps. This work will improve our ability to accurately characterize nanoscale material properties of soft materials.

Reading back stored data from Non-Volatile Memory (NVM) based devices through direct reverse engineering via scanning probe microscopy is the goal of this work. The data is stored as electrical charge in the floating gates (FG&’s) of the transistors in each memory cell. Reading these charges in the form of logic levels of “1b” and “0b” without changing the stored charges in each FG was required. Previous work has been performed on 0.35 mu;m CMOS technology node NOR Flash EEPROM [1,2]. There are no reports to date on NAND Flash devices with a 0.15 mu;m gate length. Scanning Capacitance Microscopy (SCM) with has been used, to directly to measure the programmed charges in each memory cell in a Sandisk 64MB Flash SD card memory device. The FG transistor gate length for the device measured was 0.15 mu;m, as verified by SEM cross section imaging. The device was initially programmed all over by writing a word in binary logic bits in the form of “1b” and “0b”. The sample was prepared by removing the bulk silicon substrate from the back of die, while keeping a layer of between 50 and 300 nm of thin silicon. SCM was performed by mapping the channels and active regions of the device from the backside of the die. Transistor charged values (ON/OFF or “1b/0b”) were easily distinguished in the SCM images. Reference [1] C.D. Nardi, R. Desplats, P. Perdu, C. Guérin, J.L. Gauffier, and T.B. Amundsen, Proc. 31st ISTFA, pp. 256-261, Nov. 2005. [2] C.D. Nardi, R. Desplats, P. Perdu, C. Guérin, J.L. Gauffier, and T.B. Amundsen, Proc. 32nd ISTFA, pp. 86-93, Nov. 2006.

Efficiently converting photonic to nano-plasmonic modes for localizing and enhancing optical near fields is of high interest for applications ranging from nano-optical imaging and sensing to computing. Based on extensive simulations of various “optical transformer” geometries, we propose a novel photonic-plasmonic hybrid Scanning Near-field Optical Microscopy (SNOM) probe called the “campanile” tip. These campanile tips couple the photonic to the plasmonic mode, then adiabatically compress the plasmon mode, over a broad bandwidth, which is crucial for many optical spectroscopy techniques. The confinement of the optical near field is determined by the gap size between the two antenna arms, which can be well below 10nm given the appropriate resolution of the dielectric deposition method. Based on excitation through the back of the tip similar to traditional aperture-based SNOM tips, these campanile tips are an excellent candidate for background-free nanoscale imaging and spectroscopy applications on dielectric, non-transparent substrates. We used FEM to simulate conventional aperture-based probes, the coaxial plasmonic probes, traditional apertureless SNOM tips and the state-of-the-art adiabatic-compression-type probes, and compared them all with the campanile tip geometry. The understanding of relative strengths and weaknesses of each SNOM probe geometry served as the guideline for the design of the campanile tips, resulting in their superior field coupling, enhancement and resolution capabilities. Experimentally, as a proof-of-concept, ~40nm resolution hyperspectral nanoimaging of InP nanowires is performed via excitation and collection through the campanile probes. We map the influence of local trap states on exciton energies and recombination rates, revealing optoelectronic structure along individual nanowires that is not possible to observe with any existing methods. The theory and experiments demonstrates their unique access to physiochemical properties at the length scales relevant to critical processes in nanomaterials and the campanile geometry SNOM probe thus represents a step forward in plasmonics, non-linear optics, and especially scanning near-field investigations, allowing the study of the full scope of nanostructured materials including biological samples and optoelectronic nanomaterials.

Microparticles, including nanoparticles, with a homogeneous particle size can be produced in various methods. High-vacuum or high-temperature dry processes are well known for preparing microparticles. Alternatively, wet processes are performed in liquids and have various characteristics. Wet processes, which are used under the atmospheric pressure with few restrictions on their instruments, are adaptable to various conditions. Thus, microparticles produced in wet processes are variety in their particle sizes. A unique method is known comprising preparing a water-in-oil emulsion in advance by adopting a water phase of a thick solution as a dispersion phase in a wet process, and breaking the emulsion to produce microparticles. In this study, after preparation of emulsions from aqueous ammonium nitrate and sodium nitrate solutions, the emulsions were broken to rapidly precipitate nitrates in a supersaturation state for preparation of microparticles. The configurations of the emulsion particles produced in the process were observed with an Atomic Force Microscope(SPM9700 Shimadzu Corporation; Cantilever NCHR-20 320KHz,42N/m NANO WORLD Co. Ltd.,). A fact was experimentally confirmed that an emulsion with a particle size of 20 mu;m or smaller possessed a high internal energy and existed as a homogeneous solution even if the water system was in a near supersaturation state. In the meantime, differences in physical behaviors between the water phase as the dispersion phase and the oil phase as the continuous phase were recognized by vibrating the cantilever in the dynamic mode.The difference between the dispersion and continuous phases in emulsion state and the bulk state was additionally evaluated. After the emulsions were broken in various states and energy was rapidly removed from the water phase in a supersaturation state, the sizes and configurations of the precipitated crystals were finally observed with a Laser Confocal Microscope. Especially, when two types of aqueous solutions were mixed to be used as the dispersion phase, we have confirmed that the crystals were produced as substantially equally-sized microparticles in a homogenously mixed configuration, irrespective of the differential solubilities of the substances.

Despite the evolution of scanning probe microscopy (SPM) into a powerful set of techniques that image surfaces and map their properties down to the atomic level, significant limitations in both imaging and mapping persist. Currently, typical SPM capabilities qualitatively record only one property at a time and at a fixed distance from the surface. Furthermore, the probing tip&’s apex is chemically and electronically undefined, complicating data interpretation. To overcome these limitations, we started to integrate significant extensions to existing SPM approaches. First, we extended noncontact atomic force microscopy with atomic resolution to three dimensions by adding the capability to quantify the tip-sample force fields near a surface with picometer and piconewton resolution. Next, we gained electronic information by recording the tunneling current simultaneously with the force interaction. We then moved on to study the influence of tip chemistry and asymmetry on the recorded interactions. Applications to metal oxides are shown. From this platform, we present our vision of a method capable of characterizing full atomic-scale chemical and electronic properties.

The sharp tips and piezoelectric positioning used in scanning probe microscopy are powerful tools for fabricating nanostructures as well as imaging them. Scanning probe lithography (SPL) has been very successful in a wide variety of nanofabrication methodologies ranging from positioning single atoms with a scanning tunneling microscope to patterning self-assembled monolayers in dip-pen nanolithography. Throughput is the principle barrier to widespread applicability in SPL as the serial nature of tip-based writing makes writing large area patterns prohibitively time consuming. One promising route towards performing high-throughput SPL is to utilize many tips that operate in parallel, but there are many technical challenges in creating a robust platform that retains low cost. Recently, we have developed a technique we term cantilever-free scanning probe lithography in which the cantilever of a scanning probe is replaced with an elastomeric backing layer. Using this architecture, large-scale arrays of tips can be readily fabricated at low cost. This backing layer provides compliance to each tip in a massive array and allows them to simultaneously be in contact with the surface. In addition, the optical transparency of this layer allows the array to be brought into contact and leveled with respect to a surface. We have used this cantilever-free architecture to perform large-scale lithography with arrays of soft elastomeric tips, hard silicon tips, and near-field optical apertures. Currently, these techniques write with each tip in the array in parallel. An open challenge in cantilever-free scanning probe lithography is to develop a simple method for actuating individual tips in an array, which would enable one to write truly arbitrary patterns over large areas. Several paths toward achieving large scale individual actuation have been explored and we will discuss the successes, challenges, and prospects of these massively multiplexed techniques.

Fourier-transform infrared (FTIR) spectroscopy is an established technique for characterization and recognition of inorganic, organic and biological materials by their far-field absorption spectra in the infrared (IR) fingerprint region. However, due to the diffraction limit conventional FTIR spectroscopy is unsuitable for nanoscale resolved measurements. We recently applied the principles of FTIR to scattering-type Scanning Near-field Optical Microscopy (s-SNOM) [1-4]. s-SNOM employs an externally-illuminated sharp metallic tip to create a nanoscale hot-spot at its apex which greatly enhances the near-field interaction between the probing tip and the sample. The light backscattered from the tip transmits the information about this near-field interaction to the far zone where the FTIR spectra can be recorded. The result is a novel nano-FTIR technique, which is able to perform near-field spectroscopy and imaging with nanoscale resolution. Here we demonstrate nano-FTIR with a coherent-continuum infrared light source. We show in that the method can straightforwardly determine the infrared absorption spectrum of organic samples with a spatial resolution of 20 nm. Corroborated by theory, the nano-FTIR absorption spectra correlate well with conventional FTIR absorption spectra, as experimentally demonstrated with PMMA samples. Nano-FTIR can thus make use of standard infrared databases of molecular vibrations to identify organic materials in ultra-small quantity and at ultrahigh spatial resolution. As an application example we demonstrate the identification of a nanoscale PDMS contamination on a PMMA sample [4]. We envision that nano-FTIR will become a powerful tool for chemical identification of nanostructures, for investigating local structural properties (i.e. defects, strain) of crystalline and amorphous nanostructures, as well as for non-invasive measurement of the local free-carrier concentration and mobility in doped nanostructures. [1] S. Amarie, T. Ganz, and F. Keilmann, Opt. Express 17, 21794 (2009) [2] F. Huth, M. Schnell, J. Wittborn et al, Nat. Mater. 10, 352 (2011). [3] S. Amarie, P. Zaslanky, Y. Kajihara et al, Beilstein J. Nanotechnol. 3, 312 (2012). [4] F. Huth, A. Govyadinov, S. Amarie et al, Nano Lett. In press (2012).

In SubSurface-AFM an ultrasound acoustic wave is send through the sample. This acoustic wave is Rayleigh scattered [1] at defects in the interior of the sample. In order to see where the defects are, one needs to measure the resulting amplitude and phase of the ultrasonic wave at the sample surface with a cantilever. This difficult to measure ultrasonic signal is usually obtained via mixing with another high frequency signal and reading out the non-linear low frequency difference signal. Keeping in mind the different operation and feedback modes in AFM, the important question is: "What is the correct way to measure the vibration amplitude and phase of the ultrasound wave that has travelled through the sample?" It turns out that contact mode AFM and tapping mode AFM are no options, as the particular feedback operation introduces amplitude variations and a significant phase shift during the measurement. Non-Contact AFM might be a solution, as it keeps the amplitude constant and operates with a feedback on the phase while approaching the surface. In this mode it might be even possible to directly measure the amplitude and phase of the ultrasound wave that travelled through the sample. As the tip-sample interaction is crucial for a successful measurement, we present both a numerical and experimental study of the cantilever motion in NC-AFM on a surface vibrating at ultrasonic frequencies. Understanding the measuring principle of SubSurface-AFM is the key to get 3D information of a sample. [1] G.J. Verbiest et al., Nanotechnology 23 (2012) 145704

Understanding the emergent physical phenomena at surfaces requires the capability to probe minute deviations from ideal structures, comprising order parameter fields, and exploring their coupling to electronic properties. Here, we report the studies of the chemical and electronic structure and structural distortions on the surface of La5/8Ca3/8MnO3 (001) through direct order parameter mapping by high-resolution scanning tunneling microscopy, directly visualizing order parameter fields and associated topological defects. The perovskite manganites are a hotbed of exciting physical phenomena enabled by the interplay of structural, electronic, and magnetic properties. In addition to unique physical properties, these materials possess high oxygen nonstoichiometries, affecting the physical behaviors and making them of interest for electrocatalytic applications. Many of these properties are intimately linked to small lattice/angle distortion that can significantly affect their electronic and chemical functionality. Atomically smooth and clean surfaces offer great opportunities to explore distortions because such surfaces are crucial for resolving atomic ordering using scanning tunneling microscopy. Starting from atomic resolution scanning tunneling microscopy images, we identity and refine the surface adatom locations and oxygen vacancies. From these, the local distortion angles are extracted based on the refined coordinates of surface adatoms. By this way, we can obtain angle distortion maps, in which we clearly demonstrate the boundary between two distortion domains with different distortion orientations. The distortion is shown to be sensitively affected by the STM tip bias and is lifted for positive polarities. The origins of this behavior are discussed in terms of Jahn-Teller domains and tip-induced reconstructions of adatom geometry. Overall, these studies provide an example of order parameter field mapping from high-resolution STM, opening the pathway for probing of electronic properties of topological defects and interaction between order parameter fields and structural defects. Research was supported (W.L., S.V.K.) by the U.S. Department of Energy, Basic Energy Sciences, Materials Sciences and Engineering Division. This research was conducted at the Center for Nanophase Materials Sciences, which is sponsored at Oak Ridge National Laboratory by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy.

To obtain true atomic-resolution images by frequency modulation atomic force microscopy (FM-AFM), it is required to detect short-range interaction force acting between the tip front atom and surface topmost atom. Although the minimum detectable force (Fmin) required for it depends on various conditions, it typically falls in the range of 10-100 pN. In vacuum, this condition is easily satisfied due to the high Q factor of the cantilever resonance (Q = 1,000-100,000). However, this can barely be satisfied in liquid even with the optimal operating conditions owing to the low Q factor (Q = 1-10). Such a narrow margin of the performance often leads to the low efficiency and reproducibility in experiments and practically limits the application range. The theoretical limit of Fmin in FM-AFM is ultimately determined by the cantilever parameters such as Q, resonance frequency (f0) and spring constant (k). Among them, k is often determined by the application purpose so that it is often impossible to vary it for the improvement in Fmin. The enhancement of Q-factor in liquid generally leads to an increase of k, giving little improvement in Fmin. Thus, previous efforts have mainly been focused on the enhancement of f0 to obtain a higher force sensitivity and faster time response. One of the major strategies for this is to reduce the cantilever size. By reducing the cantilever size in an appropriate manner, f0 can be increased without giving a significant change in k and Q. Although advantages of using a small cantilever have theoretically been expected, it was practically challenging at the early stage of AFM development. However, now the situation has been changed by the technical advancements. So far, applications of small cantilevers have mainly been focused on the high-speed imaging in liquid using amplitude modulation AFM (AM-AFM). These previous works demonstrated the improved time response of dynamic-mode AFM obtained by the small cantilevers. However, there have been no reports on the atomic-scale FM-AFM applications using small cantilevers with megahertz-order f0 in liquid. Therefore, it has remained unclear if there are any practical issues that may prevent such an application. In this study, we clarify the practical issues to be overcome for applying a small cantilever to atomic-scale FM-AFM experiments. We also present ways to overcome these difficulties and demonstrate stable atomic-resolution FM-AFM imaging of a cleaved mica surface in liquid using a small cantilever. In addition, we experimentally demonstrate the improvement in Fmin obtained by the small cantilever in the measurements of oscillatory hydration forces. [1] T. Fukuma, K. Onishi, N. Kobayashi, A. Matsuki and H. Asakawa, Nanotechnology 23 (2012) 135706.

High resolution AFM imaging in AC mode in air requires very careful choice of scanning parameters: free amplitude and setpoint. In conventional amplitude modulation (AM) imaging mode the decrease of amplitude during interaction of the probe with the surface comes from two different sources: dissipation and shift of the actual resonance frequency relative to the pumping frequency. We suggest the use of frequency-tracking AM mode (dissipation mode), where the actual resonance frequency is tracked (as in the frequency modulation (FM) mode), but the feedback signal for the Z-actuator comes from the amplitude channel. Thus, amplitude change of the cantilever oscillation is purely dissipative. The amplitude-vs-distance curve is monotonic and has no bistability region. There are no limitations on the setpoint choice as is the case in pure AM or FM modes. The frequency-vs-distance curve and the corresponding amplitude-vs-distance curve, recorded in dissipation mode provide clear criterion for the choice of amplitude and the imaging setpoint. The free amplitude needs to be increased until there is a clear minimum on the frequency vs distance curve. By choosing the setpoint at amplitude slightly higher than that corresponding to the frequency minimum, we limit the time the cantilever spends in the repulsive regime to a small fraction of the oscillation period, thus minimizing the invasive force exerted on the sample. Such gentle interaction allows high-resolution imaging even with relatively blunt tips. Examples of molecular resolution on functional alkane monolayers in ambient conditions, in air are presented in support of the above criterion.

Sub-harmonic excitation has been recently predicted to occur under particular conditions in amplitude modulation atomic force microscopy AM AFM[1]. These lower frequencies could have potential applications in the study of nanoscale properties. Nevertheless, there might be several mechanisms inducing sub-harmonics and, while some might be useful for compositional, chemical or material mapping, others might not [2]. Thus, here we discuss plausible mechanisms of excitation, i.e. tip trapping, grazing and forces where the onset and breakoff is distance dependent. Comparisons are made between theoretical predictions and experimental outcomes for a variety of samples. The consequences of sub-harmonics in terms of energy dissipation are also discussed. A close form solution is also provided and shown to reduce to the standard expressions, i.e. Cleveland[3, 4] and Tamayo, when sub-harmonics are inhibited [5]. Previous reports[6] showed that the standard expressions might fail in studies related to capillary interactions. In this respect, we show that this is a consequence of sub-harmonic rather than harmonics excitation. [1] Santos S, Barcons V, Verdaguer A and Chiesa M 2011 Sub-harmonic excitation in AM AFM in the presence of adsorbed water layers Journal of Applied Physics 110 114902-11 [2] Santos S, Barcons V, Gadelrab K, Guang L, Armstrong P, Stefancich M and Chiesa M 2012 A study of several mechanisms inducing sub-harmonic excitation in ambient amplitude modulation atomic force microscopy. [3] Cleveland J P, Anczykowski B, Schmid A E and Elings V B 1998 Energy dissipation in tapping-mode atomic force microscopy Applied Physics Letters 72 2613-5 [4] Tamayo J 1999 Energy dissipation in tapping-mode scanning force microscopy with low quality factors Applied Physics Letters 75 3569-71 [5] Chiesa M, Gadelrab K, Verdaguer A, Segura J J, Barcons V, Thomson H N, Phillips M A, Stefancich M and Santos S 2012 Energy dissipation due to sub-harmonic excitation in dynamic atomic force microscopy. [6] Zitzler L, Herminghaus S and Mugele F 2002 Capillary forces in tapping mode atomic force microscopy Physical Review B 66 155436-8

Recently, it has been shown that multi-frequency techniques, like dual-frequency-modulation and dual-amplitude-modulation modes can increase the observed contrast in dynamic force microscopy imaging in vacuum, air and liquids under certain conditions [1-3]. However, further studies are needed to gain more insight into the physical background for these observations. Hence, we present a systematic analysis of the atomic-scale imaging capabilities of dual-amplitude-modulation dynamic force microscopy in liquid. To study the difference in sensitivity between the 1st and 2nd eigenmode phase signals we investigate the observed atomic-scale contrasts of the mica-water interface under varying imaging conditions. For this purpose, we systematically change the main imaging parameters like the setpoint amplitude of the imaging feedback, the free oscillation amplitudes of the 1st and 2nd flexural eigenmodes, and their ratio. This allows for an in-depth analysis of the sensitivity of the 1st and 2nd eigenmode phase signals to draw conclusions regarding the underlying physical mechanisms and the interpretation of the contrast in the multi-frequency technique. [1] S. Kawai et al., Phys. Rev. Lett. 103, 220801 (2009) [2] N.F. Martinez et al., Appl. Phys. Lett. 89, 153115 (2006) [3] C.Dietz et al., Nanotechnology 22, 125708 (2011)

Bimodal atomic force microscopy is based on the simultaneous excitation of two eigenmodes of the cantilever. Bimodal AFM enables the simultaneous recording of several material properties and, at the same time, it also increases the sensitivity of the microscope. Here, the excitation of two cantilever eigenmodes in dynamic force microscopy enables the separation between topography and flexibility mapping. We have measured variations of the elastic modulus in a single antibody pentamer of 10 MPa when the probe is moved from the end of the protein arm to the central protrusion. Bimodal dynamic force microscopy enables us to perform the measurements under very small repulsive loads (30-50 pN). We also develop a model based on fractional calculus to express the frequency shift of the second eigenmode in terms of the fractional derivative of the interaction force. We show that this approximation is valid for situations in which the amplitude of the first mode is larger than the length of scale of the force, corresponding to the most common experimental case. The model allows the measurement of the effective elastic modulus and the contact radius on heterogeneous samples.

Three areas in which conventional AFM has limitations are: (i) low imaging rate, (ii) probe-sample force interaction, and (iii) the planar nature of the sample. We are developing two techniques for high-speed force microscopy. One high-speed AFM (HS AFM) technique is a DC method in which the tip is in continuous 'contact' with the specimen. This routinely allows video-rate imaging (30 frames per second, fps) and a specially developed version of this instrumentation has allowed imaging at over 1000 fps, i.e., 100,000 times faster than conventional microscopes. Damage to specimens resulting from this high-speed contact-mode imaging is surprisingly very considerably less than would be caused at normal speeds. The origin of this effect appears to lie in probe lifting off the surface by about 2 nm when travelling above a threshold speed. The other high-speed force microscope we have developed is a non-contact method based on shear-force microscopy (ShFM). In this HS ShFM, a vertically-mounted laterally-oscillating probe detects the sample surface at about 1 nm from it as a result of the change in the mechanical properties of the water confined between the probe tip and the sample. With this technique, very low forces are applied to the specimen resulting in negligible distortion. Many modes of imaging are available with this technique making it a rich areas of force microscopy development. The requirement that the sample be planar is so engrained in the operation of SPM that it is usually not recognized as a constraint. In a conventional AFM, as the tip and cantilever scan over the surface of a sample they are displaced essentially only in the direction perpendicular to the sample. It is as if the tip is only ‘seeing&’ the sample from above. For example, it is unable to ‘look&’ at the sides of structures on the sample. We have recently constructed an AFM in which the tip is the end of a nanostructure that is steered in three dimensions with five or six degrees of freedom using holographically generate optical traps. This allows the nanostructure tip to be scanned and detect the sample surface from any direction so that it is capable of imaging around 3D structures.

Present raster scan techniques are poorly matched to the instrument limitations of Atomic Force Microscopy hindering advances in high speed scanning. First, piezo hysteresis causes trace and retrace scans not to overlap such that half of the data during each frame is not utilized. Closed loop scanners do not mitigate the problem enough to enable display of both the trace and retrace in the same image. Second, the triangular fast scan wave form includes many harmonics of the fundamental line scan frequency. The bandwidth of the piezo stage must be much higher than the fundamental line scan frequency to scan without distortion. Rounding the waveform and sinusoidal scanning enable higher fundamental line scan frequencies for the same bandwidth scanner but this requires overscanning and further decreases utilization of the scan time for rendering images. We have significantly improved the frame rate by using spiral scans. Adjacent scan lines during a spiral scan have the same scan direction enabling the use of all data for generating images increasing the frame rate by at least a factor of 2.5. Also, the acceleration required for a spiral scan waveform is much lower because the scanner is continually accelerating the sample instead of only at the turn around. As a result, much faster tip velocities are possible for a specific scanner bandwidth further improving the frame rate. These high tip velocities require extremely fast Z scanners and control. We have developed a new high power amplifier design enabling a Z bandwidth well matched to the improved frame rates. Furthermore, we use advanced image processing tools such as total variation and nonlocal means inpainting to recover high-resolution images from quickly collected sparse data sets to improve temporal resolution.

With the continued development and application of nano/micro scale mechanical devices, it is increasingly important to understand nanoscale friction and wear at technically relevant sliding velocities. A high speed nanotribology approach has therefore been developed leveraging high speed SPM advances. The technique efficiently acquires friction-force curves based on a sequence of high speed images, each with incrementally lower loads. As a result, maps of the coefficient of friction, friction at zero load, and/or load for zero friction can be uniquely determined for heterogeneous surfaces. For microfabricated SiO2 islands surrounded by thiol coated Au, both the coefficient of friction, as well as the friction at zero applied force, were determined at scan velocities up to 3 mm/s. The results are within 10% of traditional, 1000 times slower AFM-based friction studies. The friction properties of mica are also reported from standard tip velocities of 2 um/sec to 10,000 times faster, 2 cm/sec. Although this quantitative friction mapping approach is not limited to high scan velocities, the results indicate that it is applicable to investigations of sliding or rolling components even at realistic speeds for MEMS, coatings, data storage devices, etc.

Research at the University of Bristol has increased the frame rate of the atomic force microscope (AFM) from one frame taking over a minute to build, to achieving video rate nano-scale imaging using high-speed contact mode AFM (HSAFM) [1]. Recent research into the cantilever dynamics [2] have led to a dramatic increase in image quality. By automatically panning the scanning window around a surface, it is possible to build up an AFM image with a lateral resolution of ten nanometres over an area in excess of 1x1cm. The total pixel count of such images can be close to a terapixel. With minor adjustments the HSAFM apparatus can map material properties such as the conductance, surface stiffness, or friction simultaneously with the surface topography. Using a conductive cantilever it is also possible to carry out nanolithography on silicon surfaces. This talk will give a brief overview of the apparatus and examples of some of the surfaces we have characterised. [1] Picco, L. M.; Dunton, P. G.; Ulcinas, A.; Engledew, D. J.; Hoshi, O.; Ushiki, T. & Miles, M. J. Nanotechnology, 2008, 19, 384018 [2] Payton, O. D.; Picco, L.; Robert, D.; Raman, A.; Homer, M. E.; Champneys, A. R. & Miles, M. J. Nanotechnology, 2012, 23, 205704

We are developing our world-leading high-speed atomic force microscope to assay the gene expression from a single cell - comprising more than 300,000 mRNA molecules - in less than 24 hours. This nanotechnology-based approach to single cell transcription profiling is hundreds of times faster and tens of thousands of times cheaper than current amplification-based techniques. The Holy Grail of transcriptional profiling is an instrument capable of routine, broad-spectrum analysis of the gene expression in single cells. AFM-based single molecule imaging provides an alternative approach to molecular recognition, opening new avenues for medical diagnostics, genetic tests and pathogen detection. Transcripts and transcript isoforms, which traditional methods like qPCR would find hard to distinguish between, can be accurately identified using AFM imaging [1]. Our HSAFM is capable of tens of images per second with nanometre resolution [2] and here we apply it to the identification of specific gene species from complex mixtures. Recent results from the development of our high-speed atomic force microscope for DNA transcription profiling are presented and the challenges faced in scanning 1 x 1 mm areas with nanometre resolution are discussed. [1] Reed, J. et al., Journal of The Royal Society Interface, March 28 (2012) [2] Picco, L. et al., Nanotechnology, 19 (2008)

The relation between cellular mechanics and disease is obviously important in cases where mechanical properties of tissues and cells are essential for cell function (like in cardiovascular diseases), and in cases where molecular mechanics is modified (e.g. in muscular dystrophies). This relation can also be important in other diseases like cancer, where its biological relevance is not so obvious, but it has been experimentally shown that cellular stiffness depends on the degree of malignancy. There are many methods that allow the detection of changes in cytoskeletal organization, manifesting in the difference in cellular deformability. One of such techniques, sensitive to changes in mechanical properties, is the atomic force microscopy (AFM), which detects pathologically changed cells via measurement of their elasticity. This offers the detection of otherwise unnoticed pathological cells, disregarded by histological analysis due to insignificant manifestations. Such measurements can also be applied to tissue sections collected from patients suffering from various diseases. The quantitative analysis of cell deformability can have a significant impact on the development of methodological approaches towards precise identification of altered cells and may lead to more effective detection of pathological changes.

A negatively charged biopolymer, the so-called glycocalyx (GC) covering the inner wall of blood vessels as well as the surface of red blood cells (RBC) prevents deleterious interactions between membrane surfaces. The GC is being destroyed by salt abuse and various inflammatory processes increasing the risk of high blood pressure, heart attack and stroke. Though fluorescence and electron microscopy can visualize the GC these approaches do not provide insight into more functional aspects at the nanoscale. Therefore we developed a method applying atomic force microscopy (AFM) for characterizing the GC of RBC in functional terms. In a first step human RBC were washed in buffer, seeded on polylysine coated glass and then immobilized by ultra-mild fixation using glutaraldehyde (about 1/50 of standard fixative concentration). This immobilization procedure stabilizes the fragile GC protein scaffold while allowing the enzyme heparinase, applied after fixation, to cleave the negatively charged GC-associated heparan sulphates. By using AFM, RBC were continuously imaged during heparinase treatment. The image before heparinase application served as the reference image while the following images, at given times after heparinase treatment, represented cells increasingly devoid of the negatively charged heparan sulphate residues. Substraction images resulted in "net images" exhibiting the GC. The "apparent" GC height was found to be between 20 and 200 nm. Since fixation of the GC protein scaffold should have prevented any physical changes in height when heparinase was applied we assume that the "net image" represents the RBC landscape of the repulsive forces between the AFM tip and the negatively charged RBC surface rather than the RBC surface morphology. In conclusion, AFM could be a useful tool for the quantitative evaluation of vascular function in man.

Recent studies indicate that astrocytes grown on nanofibrillar surfaces that approximate their native extracellular matrix environments behave differently, and in seemingly more biomimetic ways. Many details of the cell-scaffold and cell-cell interactions that may induce the biomimetic response are not presently well known. This is therefore a research area in which the nanoscale resolution capability of AFM could offer significant biomedical insights. A difficulty with AFM investigation is that the cellular edges and processes are approximately the same order in height as the background nanofibers, ~100 to 200 nm and cannot be resolved with height, deflection or phase imaging. In recent work [1], we demonstrated that this problem can be resolved by filtering and presented a novel diagnostic approach based on standard AFM section measurements that enabled knowledgeable filter selection and design. New information revealed in the clear-featured AFM images indicated that the stellate processes were longer than previously identified at 24h, in many cases extending to cell-cell interactions. It was furthermore shown that cell spreading could vary significantly as a function of environmental parameters, and that AFM images could record these variations [2]. In the present work, we compare the results of a conventional two-dimensional stellation index study of both AFM and immuno-stained fluorescent microscopy images with the results obtained using a new more three-dimensional AFM-based definition. Stellation is an important measure of cell health or pathology for several cell groups including neural, liver and pancreatic cells. [1] Volkan M. Tiryaki, Virginia M. Ayres, Adeel A. Khan, Ijaz Ahmed, David Shreiber, and Sally Meiners “Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes”, in press, Int. J. Nanomedicine (2012) [2] Volkan M. Tiryaki, Adeel A. Khan, Virginia M. Ayres, “AFM Feature Definition for Neural Cells on Nanofibrillar Tissue Scaffolds”, Scanning, e-pub ahead of print in Early View: DOI 10.1002/sca.21013 (2012)

Living neuronal cells present active mechanical structures which evolve with cellular growth and changes in the cell microenvironment. Detailed knowledge of various mechanical parameters such as cell stiffness or adhesion forces and traction stresses generated during axonal extension is essential for understanding the mechanisms that control neuronal growth and development. Here we present a combined Atomic Force Microscopy (AFM)/Fluorescence Microscopy approach for obtaining systematic, high-resolution elasticity and fluorescent maps for different types of live neuronal cells. This approach allows us to simultaneously image and apply controllable forces to neurons, and also to monitor the real time dynamics of the cell cytoskeleton. We measure how the stiffness of neurons changes both during axonal growth and upon chemical modification of the cell, and identify the cytoskeletal components most responsible for the changes in cellular elasticity. We also compare the elasticity values obtained for different types of neuronal cells and correlate these values with mechanical properties of the cell native microenvironment. Funding: NSF-CBET 1067093

Mechanical property characterization with spatial resolution of a nanometer was recognized as a grand challenge for nanotechnology a decade ago. Because atomic force microscopes (AFM) interrogate the samples mechanically and provide resolution down to the level of atoms, they are a natural candidate for nanomechanical mapping. However, factors such as tip geometry characterization, load and displacement calibration and control, and the models used to compute material properties substantially complicate the measurements. For industrial applications, throughput is an additional challenge. In the last few years, much progress has been made. A series of calibration methods for force and tip geometry have been developed. Various tip-sample interaction models were developed and validated by comparison with bulk measurements. During material property mapping, the time scale of tip-sample interaction now spans from microseconds to seconds, tip sample forces can be controlled from piconewtons to micronewtons, and spatial resolution can reach sub-nanometer. AFM has become a unique mechanical measurement tool having large dynamic range (1kPa to 100GPa in modulus) with the flexibility to integrate with other physical property characterization techniques in versatile environments. The methods of mechanical mapping have also evolved from slow force volume to multiple-frequency based dynamic measurements using TappingModeTM and contact resonance. Even more recently, high speed and real-time control of the peak force of the tip sample interaction has led to a fundamental change in AFM imaging, providing quantitative mapping of mechanical properties at unprecedented resolution. In addition, ease of use improvements and development of high speed AFM have led to faster, simpler, and more quantitative SEM like operation with the AFM. This presentation will review this recent progress, providing examples from a wide range of fields that demonstrate the high resolution and wide dynamic range of the measurements, as well as the speed and ease with which they were obtained.

Biological systems exhibit rich molecular compositions and mechanical properties at the molecular, cellular, and organismic levels. Beyond the variety in length scales, these characteristics are dynamic over broad range of time scales. We will present atomic force microscopy based experimental techniques that can access forces and displacements on the nanoscale with a temporal resolution around one microsecond. Based on this nanomechanical platform, we have developed a single-molecule biophysical assay to probe biomolecular interactions with short lifetimes and a high-resolution mechanical imaging method for probing live eukaryotic cells. Our approach is based on T-shaped cantilevers with tips placed offset from the center of the cantilever. In addition to their ability to make rapid, quantitative measurements, these cantilevers offer important advantages for measurements in liquid, because they can isolate tip-sample interactions from large viscous drag forces. While viscous drag forces act evenly on both arms of the T-shaped cantilever, tip-sample forces act asymmetrically.

We describe a new quantitative method to study viscoelastic (frequency-dependent) properties of soft materials, such as biomaterials, cells, tissues, polymers, and nanocomposites, at the nanoscale. The method is based on the use of AFM, and allows analyzing viscoelastic properties of materials with a nanoscale probe much faster (>100x) and with higher lateral resolution (>100x times) compared to the existing nanoindentation technique. The main idea of the method is to measure multiple frequencies at the same time, not sequentially as done in the existing nanoindenters. This can obviously accelerate the measurements for any material, but it brings a true breakthrough for soft materials. Besides much faster measurements, a substantially higher spatial resolution is attained because there is no need in waiting for slow relaxation of soft materials (called “creep”, a slow deepening of the indenter into material under constant load). The existing indenters have to wait until the contact area between the indenter probe and surface stabiles. In our approach, the measurements are fast enough to avoid waiting for the creep relaxation. It allows keeping the area of probe-surface contact small or spatial resolution high. In addition, our technique allows for testing the linearity of strain-stress relation at the nanoscale while doing the measurements (such linearity information is paramount for proper calculation of the rigidity modulus).

Contact-resonance atomic force microscopy (CR-AFM) constitutes a highly promising upcoming scanning probe microscopy technique, that allows to quantify mechanical properties of samples in surface proximity with nanometer resolution. Basically, a cantilever is brought into direct contact with the sample surface with a predefined static load, while broad band longitudinal acoustic waves are fed into the sample by an ultrasound transducer and excite the cantilever-sample system to vibrations. From the cantilever eigenfrequencies during material contact information on the mechanical surface properties, viz. the indendation modulus, is accessible with nanometer resolution. Within the present contribution we first review the fundamentals of this technique, report about our implementation into a standard atomic force microscope (AFM) and address model assumptions that are employed within finite element calculations to extract indentation moduli from experimental data. The capabilities of the CR-AFM technique particularly become obvious for samples with nanoscale mechanical heterogeneities at the surface. One particularly interesting example are single crystalline Ni-Mn-Ga ferromangetic shape memory alloy thin film, where we are able to resolve mechanical contrast by individual twin boundaries within the martensite phase [1]. Comparing our CR-AFM measurement directly to ab-initio calculations on mechanical properties in this alloy, we are able to interpret the physics occuring during CR-AFM measurment of this alloy. Within this context we also discuss the limitations of the CR-AFM technique, which need to be carefully addressed in particular for surfaces with topographic features with lateral dimensions comparable to the radius of the AFM tip. Future prospects and challenges of CR-AFM, including sub-surface imaging, are also discussed. [1] A.M. Jakob, M. Müller, B. Rauschenbach and S.G. Mayr, New Journal of Physics, 14, 033029 (2012)

Nanoscale characterization of viscoelastic properties is essential for development of advanced polymers and biomaterials. To address this need, a number of atomic force microscopy (AFM) methods have been developed to provide data on storage modulus Eprime;, loss modulus EPrime;, and loss tangent (tan δ). Such methods include force modulation microscopy (FMM), viscoelastic contact resonance force microscopy (CR-FM), and phase/amplitude imaging in intermittent contact mode. In this work, we present results with several established and emerging AFM methods to evaluate their potential for accurate, nanoscale viscoelastic mapping. We first describe each method&’s measurement and analysis concepts and then present experimental results for several model materials. These materials cover a significant range of Eprime; (approximately 0.5 GPa to 5 GPa) and tan δ and include polymers such as polyurethane with different crosslink densities, polypropylene, and high density polyethylene. The results with AFM methods are compared to those obtained with complementary techniques on larger length scales, for instance macro- and microscale dynamic mechanical analysis (DMA). Results are compared taking into consideration the characteristic frequencies of each method and the frequency dependence of viscoelastic properties. In this way, we evaluate the applicability of AFM methods for reliable viscoelastic mapping and identify potential measurement improvements, for instance by combining quasistatic and dynamic techniques. Our results will help to advance the state of the art in quantitative measurements of nanoscale viscoelastic properties.

As a protein-mineral composite, bone combines the optimal properties of both components and provides both rigidity and resistance against fracture with its unique hierarchical structure and the interactions between different constituents. The unique hierarchical structure of bone is a subject of extensive investigations at many length scales both through experiments and modeling. Scanning probe microscopy as well as nanoindentation experiments provide unique insight into nanomechanical behavior of bone. Our group&’s multiscale modeling work demonstrated that the mechanical behavior of collagen is significantly influenced by collagen-mineral interaction as well as collagen-water-mineral. Chemical pretreatment has been a part of the standard sample preparation procedure in spectroscopic study and the study of nanomechanical properties of bone but has been known to influence molecular structures. Several studies have shown that chemical pretreatment might affect both the interactions among the components of bone and its mechanical properties. Thus we have conducted experiments on undisturbed bone using unique experiments in scanning probe microscopy, nanomechanical testing and infrared spectroscopy. We have also conducted photoacoustic-Fourier transform infrared spectroscopy (PA-FTIR) experiments to investigate the orientational differences in molecular structure of human bone. These insitu spectroscopic investigations reveal orientational differences in stoichiometry of hydroxyapatite. Further, comprehensive dynamic and static nanomechanical testing including nanomodulus mapping was conducted over the two directions in the human bone: transverse and longitudinal. Consistent with the photoacoustic experiments, differences were observed between dynamic response from transverse and longitudinal sections of human bone. In addition, stiffness of collagen fibrils and their inherent frequency characteristics were obtained in transverse section. Microstructural differences were ascertained using imaging capabilities of scanning probe and scanning electron microscopies. These differences could be attributed to the structural differences in the longitudinal and transverse directions consistent with the complex hierarchical organization of human bone. These results can provide an insight into nanoscale properties and relationships to macroscale response of bone.

Many molecules exhibit unique vibrational peaks free from overtones in the mid-infrared region of the spectrum. Resonant excitation of the molecules adsorbed on surfaces using infrared light can result in significant heat generation as a result of nonradiative processes. If the surface is that of a microfabricated bimaterial cantilever, then the thermal changes due to resonant excitation of the molecules can be observed as cantilever bending. Utilizing the thermal sensitivity of bimaterial cantilevers in mid-infrared spectroscopy opens up new possibilities for molecular recognition of picogram (pg) amounts adsorbed molecules. In this technique, known as photothermal cantilever deflection spectroscopy (PCDS), measurement of the cantilever bending as a function of the illumination wavelength, replicates the vibrational spectra of the adsorbed molecules. The main advantage of PCDS is its ability to detect physisorbed molecules with picogram sensitivity without using any immobilized chemical interfaces. In addition to serving as a high selectivity platform for cantilever-based chemical and biological sensing, this technique can also be used in atomic force microscopy for molecular recognition. In this case, the imaging cantilever probe, which is in contact with the sample, is excited acoustically by exciting the sample. By illuminating the sample with infrared radiation using for example total internal reflection in a prism, the response of the material causes phase changes in the cantilever resonance, which in turn provides a wavelength dependent contrast.

Scanning Torsional Thermal Microscopy (STTM) is relatively new scanning thermal microscopy approach. STTM offers superior thermal and spatial resolution without the need of external electronics. Furthermore, the probes are highly economical due to the simplicity of the cantilever design. STTM operates by using a cantilever that employs a thermal bimorph geometry that twists instead of bends upon heating. This is achieved by applying a thermal coating to half of the top side of the cantilever and the opposite half of the bottom side of the cantilever. This twisting motion does not interfere with the topographical scanning atomic force microscope, but instead uses the lateral deflection signal of the photodiode. Here, we present recent progress in STTM, including performance of second generation probes.

Optical properties of micro/nano materials are important for many applications in biology, optoelectronics, and energy. A method is described to directly measure the quantitative absorptance spectrum of micro/nano-sized structures using Fourier Transform Spectroscopy. The measurement technique combines the optomechanical cantilever probe with a modulated broadband light source from an interferometer for spectroscopic measurements of objects. Previous studies have demonstrated the use of bilayer (or multi-layer) cantilevers as highly sensitive heat flux sensors with the capability of resolving power as small as ~4 pW. Fourier Transform Spectroscopy is a well-established method to measure broadband spectra with significant advantages over conventional dispersive spectrometers such as a higher power throughput and signal-to-noise ratio for a given measurement time. By integrating a bilayer cantilever probe with a Fourier Transform Infrared (FTIR) spectroscopy system, the new platform is capable of measuring broadband absorptance spectra from 3 to 18 microns directly and quantitatively with an enhanced sensitivity that enables the characterization of micro- and nanometer-sized samples, which cannot be achieved by using conventional spectroscopic techniques. Acknowledgments: This work is supported by DOE BES grant No. DE-FG02-02ER45977 (W.C.H. and B.B.), DOD MURI via UIUC FA9550-08-1-0407 (J.T., B.B., and G.C.),.) and the National Research Fund, Luxembourg grant No. 893874 (B.R.B.)

We report variable temperature nanomechanical spectroscopy using wide-bandwidth force excitation of a self-heated AFM cantilever. The technique is based on the Lorentz force created by the interaction of a current flowing through the AFM cantilever with fields from a permanent magnet focused near the cantilever. By oscillating the current through the self-heating cantilever, the tip-sample force can be modulated at frequencies from DC to the MHz regime. The Lorentz drive scheme provides clean mechanical excitation of the cantilever and avoids excitation of parasitic resonances that are common in standard piezo-based cantilever drives. The low-noise and adjustable force drive scheme enables high quality excitation over a large range of frequencies including both flexural and torsional contact resonances of the cantilever. The measured contact resonance spectra contain information about the relative tip-sample forces in both vertical and lateral directions. We analyze these mechanical spectra using beam mechanics models to extract mechanical properties of the sample, including normal and lateral contact stiffnesses and damping. Imaging at selected drive frequencies or actively tracking a specific contact resonance while scanning can result in high-contrast images that highlight sample compositional differences in heterogeneous materials. By selecting the drive frequency to match a flexural or torsional resonance, we can choose whether to probe primarily normal or lateral sample properties. Further, by controlling the tip temperature we can also rapidly probe the temperature dependence of the nanomechanical properties of the sample surface. We have applied this technique to a variety of polymeric formulations and will show results on samples of asphalt, multicomponent polymer blends, and nanocomposites.

Cantilevered scanning probes have been very successful due to their high mechanical bandwi