Alexei Gruverman University of Nebraska-Lincoln
Igor Sokolov Clarkson University
Zoya Leonenko University of Waterloo
Masamichi Fujihira Tokyo Institute of Technology
U1: Probing Mechanical Properties of Biomaterials
Tuesday PM, April 06, 2010
Room 3000 (Moscone West)
10:00 AM - U1.2
Effects of DSS Peptide on Nano-mechanical Behaviors of Demineralized Human Enamel after Different Mineralization Treatments.
Chia-Chan Hsu 1 , Hsiu-Ying Chung 2 , Elizabeth Hagerman 3 , Wenyuan Shi 4 , Jenn-Ming Yang 1 , Ben Wu 1 3 4 Show Abstract
1 Materials Science and Engineering, University of California Los Angeles, Los Angeles, California, United States, 2 Materials Science and Engineering, Feng Chia University, Taichung, 407, Taiwan, 3 Bioengineering, University of California, Los Angeles, Los Angeles, California, United States, 4 School of Dentistry, University of California, Los Angeles, Los Angeles, California, United States
Aspartate-serine-serine (DSS) repeats are abundant in naturally occurring proteins that are critical for tooth formation. We recently developed octuplet repeats of aspartate-serine-serine peptides to promote hydroxyapatite nucleation from free ions. In this study, we report a possible role of DSS in promoting mineral deposition onto human enamel. Nanoindentation results show that remineralization treatments effectively improve the mechanical and elastic properties for demineralized enamel. The difference in surface roughness and DSS-8 binding affinity among the native and demineralized enamel surfaces may account for this result. The hardness and elastic modulus for the demineralized enamel remineralized with DSS peptide are higher than those remineralized without the addition of DSS peptide. Moreover, both the type and morphology of the newly grown minerals dominate the resulting mechanical and elastic properties. The demineralized enamel remineralized with DSS peptide in 2x SBF solution possesses the highest hardness and elastic modulus. This is most likely, a consequence of the uniform calcium phosphate carbonate and HA formation, which creates a smooth surface.
10:15 AM - U1.3
Nanoscale Measurement of Carbon Nanofiber Elasticity.
Hanna Nilsson 1 , Sandeep Kaur 1 , Geetha Dholakia 1 , Carmen Lilley 2 , Cary Yang 1 Show Abstract
1 Center for Nanostructures, Santa Clara University, Santa Clara, California, United States, 2 Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, Illinois, United States
Carbon nanostructures such as nanotubes and nanofibers (CNF) have been studied extensively in the past decade. However, detailed research on the elastic properties of CNFs in the nanoscale is required to fully understand the relationship between the CNF nanostructure and influence of the substrate or support, which contributes to the overall mechanical properties of each CNF. Force spectroscopy using an atomic force microscope is a powerful tool that can reveal the relationship between a nanofiber’s local structure and its elasticity . In this study we report detailed analysis of mechanical properties of carbon nanofibers using force measurements obtained for various configurations of the nanofiber. Force-distance (F-d) relations are determined for CNFs resting on bare substrates, suspended between gold contact pads, and in configurations where they are completely encapsulated vertically in a SiO2 matrix. The results are compared with reference materials such as ceramic, gold, SiO2, and silicon nitride. The measured F-d represents the cantilever deflection vs piezo extension. Van der Waal’s forces between the nanofiber and the tip as well as local variations in the elasticity of the nanofiber both contribute to the cantilever deflection. When the measurements are performed in the contact mode, where the spring constant of the tip is higher than the softer nanofibers, the elastic modulus as a function of the nanofiber diameter (50 to 250 nm) is obtained from the slope of the force-distance curves. Measurements on CNFs suspended over gold contact pads yield an elastic modulus in the range of 100-200 GPa. Interestingly, in comparison to the nanofibers, the reference gold sample shows a larger attractive van der Waals dip, while the oxide surface has a steeper force response due to its larger stiffness. Preliminary analysis of the curves from the encapsulated CNFs also shows a steeper response. Experiments are carried out to probe along the length of a suspended fiber to investigate local variations of the elastic modulus as a function of the distance between the measurement point and the point of suspension. The results from this study will elucidate the relationship between the CNF nanostructure in the test configuration and its mechanical properties.. Lawrence, J.; Berhan, L.; Nadarajah, A. “Elastic Properties and Morphology of Individual Carbon Nanofibers”. ACS Nano, Vol. 2, No. 6, 2008.
10:30 AM - U1.4
Mechanical Behavior of Human Osteoblast Cells and Osteoblast-substrate Interactions Using Nanoindentation.
Rohit Khanna 1 , Kalpana Katti 1 , Dinesh Katti 1 Show Abstract
1 Civil Engineering, North Dakota State University, Fargo, North Dakota, United States
Biological cells are far more complex in their structure and functionality as compared to engineering materials such as metals, ceramics, polymers and composites. Mechanical behavior of various biological tissues and engineering materials has been studied using nanoindentation technique. However, live cell nanoindentation and cell-substrate mechanical properties of osteoblast cells on synthetic biodegradable materials have not been studied using the nanoindentation technique. Mechanics of cells and cell-substrate evolves over time during a series of cellular processes occurring as cells and substrates interact during cell adhesion, proliferation, differentiation as scaffold degrades. The nanomechanical response of cells during these cellular responses and that of degrading substrate need to be understood in order to evaluate the tissue mechanics. This work focuses on design and development of in vitro nanoindentation technique to study the mechanical behavior of live cells under conditions as similar to the natural environment. In this work, biocompatible and biodegradable chitosan-polygalaturonic acid-nanodydroxyapatite (Chi-PgA-HAP) nanocomposites and non-degradable tissue culture polystyrene (TCPS) substrates were used. Nanoindentation tests were carried out on dry, wet and cell-seeded substrates using displacement-controlled nanoindentation technique in a hydrated environment (cell culture fluid; 37oC). Atomic force microscopy imaging indicates osteoblast cells take the shape of degrading Chi-PgA-HAP substrate as compared to flat morphology observed on TCPS substrate. Mechanical behavior of cell and cell-substrate interactions will be discussed in the presentation.
11:15 AM - **U1.5
Quantitative Mechanical Mapping of Biomolecules in Fluid.
Chanmin Su 1 Show Abstract
1 , Veeco Instruments, Santa Barbara, California, United States
Though atomic force microscopy (AFM) interrogates biological materials through mechanical interactions, achieving quantitative mechanical information such as modulus and adhesion at high resolution has been challenging task. This presentation reports progress using peak force controlled AFM imaging to map mechanical properties of a broad range of samples including cells, collagen, protein membranes and DNA. The peak interaction force, ranging from 15 pN to 10 μN, was controlled precisely similar to triggered force volume but with over 3 orders of magnitude higher efficiency and sensitivity of force level. The interaction force control at picoNewton levels yields high resolution images while other mechanical data are recorded simultaneously. Using direct peak force control, imaging in fluid also becomes far easier by eliminating many control parameters and the need to tune the cantilever resonance. Quantitative mechanical data are derived from the measured force interactions by fitting an appropriate indentation model along with accurate calibration of cantilever spring constant and tip radius. For elastic modulus measurements, a set of bench mark samples from 0.7 MPa to 70 GPa with well controlled properties were used to verify the accuracy. Data from bacteriorhodopsin membrane, lambda DNA, collagen and living cancer cells will be discussed as examples of the new peak force controlled mapping method.
11:45 AM - U1.6
Multiple Imaging Regimes of Carbon Nanotube Tipped Probes in Tapping Mode Atomic Force Microscopy.
Mark Strus 1 2 , Arvind Raman 1 Show Abstract
1 Mechanical Engineering, Purdue University, West Lafayette, Indiana, United States, 2 Materials Relability, National Institute of Standards and Technology, Boulder, CO, Colorado, United States
Carbon nanotubes (CNTs) have emerged as ideal tips for high-resolution dynamic Atomic Force Microscopy (dAFM) and oxidation lithography because they are sharp, flexible, conducting, non-reactive, and wear-resistant. CNT tips can also be readily functionalized with biomolecules, coated with hydrophilic chemicals for imaging in liquid, and may be especially well-suited for imaging biological materials as they tend to bend, buckle, or slide to absorb energy when the tip-sample interaction forces become large, thus preventing damage to the sample. This research shows that CNT tipped AFM cantilevers can take on several distinct oscillation states in tapping mode AFM, including: non-contact attractive regime oscillation, intermittent contact with CNT slipping or pinning, and permanent contact with the CNT in point- or line-contact with the surface while the cantilever oscillates with large amplitudes. Continuum CNT models with van der Waals interactions capable of large deformations are used with either perfect slip or perfect pin boundary conditions to explain static force-distance measurements and identify these CNT AFM probe oscillation states. We also present a method to identify if the CNT slips laterally on the surface or remains pinned in the intermittent contact regime by comparing phase contrast images and energy dissipation on graphite, graphene oxide, and silicon oxide surfaces. Because each of these states represents fundamentally different origins of CNT-surface interactions, a clear understanding of the different oscillation regimes may provide new opportunities for improved biochemical applications, nanolithography, and CNT nanodevices, while CNT slipping can potentially lead to topographical errors, improved phase contrast, and novel friction-mapping AFM modes.
12:00 PM - U1.7
Imaging the Elasticity of Biopolymer Networks Within Polyelectrolyte Complexes Using Ultrasonic Force Microscopy.
Oscar Valdes 1 , Teresa Cuberes 2 Show Abstract
1 Center of Biomaterials, University of La Habana, La Habana Cuba, 2 Laboratory of Nanotechnology , University of Castilla-La Mancha, Almaden Spain
Ultrasonic Force Microscopy (UFM) belongs to a novel family of Scanning Probe Microscopy techniques based on the use of AFM with ultrasound excitation, initially developed to implement a near-field probe to emulate the Acoustic Microscope with nanoscale resolution . The UFM procedure is based on the mechanical-diode effect [1, 2] which occurs due to the net force acting upon the AFM cantilever tip during each ultrasonic cycle because of the non-linearity of the tip-sample interaction force. A mechanical-diode response can also be detected in liquid environments . UFM has been successfully applied to investigate the nanoscale distribution of elastic phases in polymer gels in ambient conditions , and in lipid bilayers in aqueous solution . A unique advantage of the ultrasonic-AFM techniques is their ability to map nanoscale subsurface elastic inhomogeneities [1, 5].In this contribution, we have applied UFM at polyelectrolyte complexes formed from Poly(acryloxyethyl-trimethylammonium chloride-co-2-hydroxyethyl methacrylate) [poly(Q-co-H)] / sodium alginate gel (Ca2+) [AlgNa] / poly-l-lysine [PLL] films, prepared on mica surfaces . The UFM images reveal nanoscale variations in elasticity that can be explained taking into account the bonding structure in the encapsulated AlgNa biopolymer network. Alginate is obtained from the Phaeophyceae brown seaweeds a as linear non-branched polymer containing 1, 4 - β - D-mannuronic acid (M) and 1, 4 - α - L - guluronic acid (G) residues. It gelifies in the presence of bivalent ions such as Ca2+. In the UFM contrast, guluronic residues with Ca2+ crosslinks in the alginate gel are identified as stiffer regions than the adjacent polymer formed by mannuronic or unreacted guluronic parts. Our results demonstrate the potential of UFM to get insight into the elastic behavior of encapsulated bionetworks and explore their promising applications in biomedical engineering. M. T. Cuberes in “Applied Scanning Probe Methods XI”, B. Bhushan, H. Fuchs (ed.), Springer-Verlag Berlin Heidelberg (2009) 39-71.  J. J. Martínez and M. T. Cuberes, in Nanoscale Tribology—Impact for Materials and Devices, edited by Y. Ando, R. Bennewitz, R.W. Carpick, W.G. Sawyer, Mater. Res. Soc. Symp. Proc. Volume 1085E, Warrendale, PA (2008).  M. T. Cuberes, J. of Physics: Conf. Ser., 100 (2008) 052014.  B. Talavera, J. J. Martínez, F. Santiago, and M. T. Cuberes, in Nanoscale Tribology—Impact for Materials and Devices, edited by Y. Ando, R. Bennewitz, R.W. Carpick, W.G. Sawyer, Mater. Res. Soc. Symp. Proc. Volume 1085E, Warrendale, PA (2008).  L. Tetard, A. Passian, et al. Nature Nanotechnology 3 (2008) 501-505; M. T. Cuberes et al., J. Phys. D: Appl. Phys 33 (2000) 2347-2355.  O. Valdés and M. T. Cuberes, J. Biomed. Nanotechnol. (in press)
12:15 PM - U1.8
Statistics of Single Cell Mechanics Investigated by Atomic Force Microscopy.
Takaharu Okajima 1 , Shinichiro Hiratsuka 1 , Yusuke Mizutani 1 , Masahiro Tsuchiya 1 , Hiroshi Tokumoto 1 , Koichi Kawahara 1 Show Abstract
1 , Hokkaido University, Sapporo Japan
Living cells respond to external mechanical stimuli and express their functions, so that it is crucial to understand the viscoelastic properties of single cells under the physiological conditions. In this study, we developed a new AFM technique to measure the mechanical properties of a large number of live cells for a short time. In this technique, the live cells were arranged and cultured in a micro-fabricated glass substrate so that the AFM force measurements could be examined in many different cells automatically. In the experiments, firstly we could obtain the number distribution of live cells, consisting of more than 300 cells, not only in the stably adhesive state but also in their transient states such as early stage of adhesion and just after exposing agents. We found that the number distribution of elastic modulus of cells in both states followed a log-normal distribution and that the log-normal distribution were gradually shifted without changing the deviation as the adhesive time and the amount of agents were increased. Moreover the number distributions of the viscoelastic properties of a large number of cells have been successfully measured by using the force modulation mode and stress and creep relaxations. Since in general the live cells exhibit temporal fluctuations as well as individual differences in their physiological conditions, the present technique is useful to study the statistics of cells at single cell level.
12:30 PM - U1.9
Mapping of the Solid-liquid Adhesion Energy With Sub-nanometer Resolution.
Kislon Voitchovsky 1 , Jeffrey Kuna 1 , Sonia Antoranz Contera 2 , Francesco Stellacci 1 Show Abstract
1 , Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 , University of Oxford, Oxford United Kingdom
Solid-liquid interfaces play a fundamental role in phenomena such as surface electrochemistry, catalysis, wetting, self-assembly, and protein functions. However, little is known about their molecular structure, mostly due to the lack of experimental techniques able to resolve and quantify interfacial energy with sufficient lateral resolution. Here we present a new approach based on amplitude modulation atomic force microscopy to investigate solid-liquid interfaces. Our approach, possible with a commercial instrument, exploits the structured liquid layers close to the solid to enhance lateral resolution and gain quantitative insight. We present atomic- or molecular-level resolution images of the interface over a broad range of samples. We propose a model to explain the mechanism dominating the image formation and show that the energy dissipated during acquisition can be directly related to the interfacial adhesion energy between the sample and the solvent. Consequently, we present sub-nanometer resolution maps of interfacial energy on many samples, some containing defects. We believe that our topography images and interfacial energy maps, not achievable before, could substantially contribute to the understanding of interfaces.
12:45 PM - U1.10
Dynamic Force Spectroscopy: Analysis of Reversible Two-state Systems.
Raymond Friddle 1 , Peter Talkner 2 , James De Yoreo 1 Show Abstract
1 , Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Institute for Physics, University of Augsburg, Augsburg Germany
The dissociation of inter- and intra-molecular bonds by force is a process that occurs regularly in biological machinery, and forcing such transitions in a controlled environment has emerged as a modern practice for studies of the physical principles of bond lifetimes and protein unfolding. We are using this technique in an effort to understand the fundamental physics of the directed assembly of virus “building-blocks” on chemically patterned templates. It is commonly assumed that force-driven dissociation is irreversible, which leads to the analysis of first-passage statistics and results in simple analytical results for the distribution and moments of the transition force. However, the irreversible model is a first-order approximation which is only valid very far from equilibrium, or under specific irreversible circumstances. Furthermore, the irreversible model has led many to conclude that force spectra that deviate from linearity unequivocally represent multiple energy barriers along the intermolecular reaction coordinate. We show that irreversible first-passage analysis, which fails for two-state systems, can be replaced by analyzing the conditional single-passage time between the two states. We find simple solutions for the forward and time-reversed distributions of the transition force, and the isothermal work, which analytically satisfy the fluctuation theorem. We also define how stochastic force trajectories should be measured when multiple forward-reverse events occur. By accounting for reversibility, we show that both the distribution and the first moment of the rupture force significantly differ from the irreversible model and clearly connect with the equilibrium regime. We find that the resulting spectrum of rupture forces is not monotonic with log of the loading rate, but follows at least two major regimes – a linear-response and a dynamic response – with the linear regime tending to the equilibrium free energy change. We validate our analytical results with simulations and experimental data of rupturing Histidine/Ni-NTA complexes, virus/substrate adhesion, and RNA unfolding.
U2: Biomolecules and Bioassemblies I
Tuesday PM, April 06, 2010
Room 3000 (Moscone West)
3:00 PM - U2.2
Automated Processing for High-throughput Analysis of Force Maps in the Study of Supported Lipid Bilayer Membranes by AFM.
Ruby May Sullan 1 2 , James Li 2 , Gilbert Walker 2 , Shan Zou 1 Show Abstract
1 Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, Ontario, Canada, 2 Chemistry, University of Toronto, Toronto, Ontario, Canada
The use of AFM force maps in studying phase segregation of multicomponent lipid membrane systems is a valuable complement to other biophysical techniques such as imaging and spectroscopy, as it provides unprecedented insight into lipid membrane mechanical properties and functions especially due to its ability to observe nanoscale system response as a function of spatial position. However, one is faced with the subsequent (lengthy) analysis following the collection of thousands of AFM force curves. To this end, a customized code was developed to facilitate with the automated high-throughput processing of these force curves to generate force maps and statistics. This tool has been successfully utilized in the study of a number of systems, including the dioleoylphosphatidylcholine/ egg sphingomyelin/ cholesterol (DEC) system. We demonstrated the direct correlation of the nanomechanical property and self-organized structure exhibited in such a phase-segregated supported lipid bilayer system by examining AFM tip-induced membrane rupture (breakthrough) events over different regions of interest. The intrinsic breakthrough forces, along with elastic moduli, adhesion forces, and indentation of the different phases in the bilayers were systematically determined on the nanometer scale and represented as force maps.
3:15 PM - U2.3
Atomic Force Microscope Imaging and Force-mapping of Phase-Segregated Lipid Bilayers on Gold and Mica Surfaces.
Shell Ip 1 , James Li 1 , Gilbert Walker 1 Show Abstract
1 Chemistry, University of Toronto, Toronto, Ontario, Canada
Planar supported lipid bilayers (SLBs) are often studied as model cell membranes because they are accessible to a variety of surface-analytic techniques that have contributed to identifying membrane lateral organization and heterogeneity as fundamentally important to a range of cellular functions and diseases. The relevance of surface plasmon resonance (SPR) - a highly surface-sensitive optical analytic technique - to studying membrane function would be greatly improved by the ability to spontaneously form bilayers of model, raft-forming lipid mixtures, on noble-metal surfaces required for SPR. We report the formation of raft-forming dioleoylphosphatidylcholine (DOPC)/sphingomyelin/cholesterol bilayers on ultra-flat gold by spontaneous fusion of unilamellar vesicles, without the use of charged, or chemically modified headgroups. The bilayers phase segregate into liquid-ordered (lo) and liquid-disordered (ld) domains, as observed by atomic force microscopy height and phase imaging, using a magnetically driven cantilever in intermittent contact mode. Furthermore, the mechanical properties of the bilayer were characterized by force-indentation maps, where each force curve is analyzed offline using in-house automated algorithms that provide access to additional image contrast mechanisms and statistics. The results were compared to the same lipid membrane system formed on mica. We found good agreement between the mica and gold supported bilayers in terms of the magnitudes of the apparent Young’s moduli of the two phases.
3:30 PM - U2.4
Probing Membrane Electrostatics by Atomic Force Microscopy.
Yi Yang 1 , Kathryn Mayer 1 , Jason Hafner 1 Show Abstract
1 Physics & Astronomy, Rice University, Houston, Texas, United States
The atomic force microscope (AFM) is sensitive to electric double layer interactions in electrolyte solutions, but provides only a qualitative view of interfacial electrostatics. We have fully characterized silicon nitride probe tips and other experimental parameters to allow a quantitative electrostatic analysis by AFM, and we have tested the validity of a simple analytical force expression through numerical simulations. As a test sample, we have measured the effective surface charge density of supported zwitterionic dioleoylphosphatidylcholine membranes with a variable fraction of anionic dioleoylphosphatidylserine. The resulting surface charge density and surface potential values are in quantitative agreement with those predicted by the Gouy-Chapman-Stern model of membrane charge regulation, but only when the numerical analysis is employed. In addition, we demonstrate that the AFM can detect double layer forces at a separation of several screening lengths, and that the probe only perturbs the membrane surface potential by approximately 2%. The high force sensitivity of the AFM has enabled the direct observation of an enigmatic electrostatic membrane property: the membrane dipole potential. The electrostatic properties of biological membranes are usually described by three parameters: the transmembrane potential, the membrane surface potential, and the membrane dipole potential. The first two are well characterized in terms of their magnitudes and biological effects. The dipole potential, however, is not well characterized. Various methods to measure the membrane dipole potential indirectly yield different values, and there is not even agreement on the source of the membrane dipole moment. This ambiguity impedes investigations into the biological effects of the membrane dipole moment, which should be substantial considering the large interfacial fields with which it is associated. Electrostatic analysis of phosphatidylcholine lipid membranes with the atomic force microscope reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipids. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internal membrane dipole potential. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of 1275 mV for supported phosphatidylcholine membranes. This new ability to quantitatively measure the membrane dipole moment in a noninvasive manner with nanometer scale spatial resolution will be useful in identifying the biological effects of the dipole potential.
4:15 PM - **U2.5
Understanding Functional Amyloid Using Atomic Force Microscopy.
Suzi Jarvis 1 Show Abstract
1 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin Ireland
Proteinaceous fibre or filament structures such as actin, microtubules and collagen are ubiquitous in nature, providing structural support both inside and outside the cell. Recently another fibre-like structure of aggregated protein has come under scrutiny for its natural and technological application as a mechanically functional structure [1, 2]. What makes this particular structure unique and very different from those studied previously is its lack of specificity in terms of amino-acid composition or sequence. These fibres, known as amyloid or amyloid-like fibrils, share a common crossed β-sheet core structure, despite the fact that the fibrils can be formed from an apparently boundless range of amino-acid sequences and lengths under appropriate conditions. Atomic force microscopy represents an ideal tool to characterise amyloid structures under physiological conditions both in terms of their nanoscale mechanical properties and their surface structure. To date, most structural investigation of amyloid fibrils has been motivated by their close association with a range of human diseases and has primarily involved the study of fibrils formed in vitro from synthesized peptides of the specific amino-acid sequences central to disease pathology . The structural investigation of extracted ex vivo fibrils is usually hindered by the presence of non-fibrillar glycoproteins and glycosaminoglycans and the resulting damage induced in the fibrils during separation and purification processes . Thus, although the generic cross β-sheet structure common to both in vitro and ex vivo fibrils has been well-studied, potential differences between fibrils formed in the two very different environments has not been fully explored. Here by investigating two physiological amyloid fibril systems that can occur in isolation rather than in a heterogeneous plaque, we are able to elucidate for the first time the mechanical characteristics of these ex vivo fibrils and make a direct comparison with amyloid-like fibril samples formed in vitro. These studies have potential consequences for the application of amyloid fibrils in biomaterials.1. Mostaert, A. S. & Jarvis, S. P. Beneficial characteristics of mechanically functional amyloid fibrils evolutionarily preserved in natural adhesives. Nanotechnology 18, 044010 (2007).2. Fukuma, T., Mostaert, A. S. & Jarvis, S. P. Explanation for the mechanical strength of amyloid fibrils. Tribol. Lett. 22, 233-237 (2006).3. Makin, O. S. & Serpell, L. C. Structures for amyloid fibrils. FEBS J. 272, 5950-5961 (2005).4. Jimenez, J. L., Tennent, G., Pepys, M. & Saibil, H. R. Structural diversity of ex vivo amyloid fibrils studied by cryo-electron microscopy. J. Mol. Biol. 311, 241-247 (2001).
4:45 PM - U2.6
Atomic Force Microscopy Studies of Protein Structure and Activity on Biomaterial Surfaces.
Christopher Siedlecki 1 2 , Pranav Soman 2 , Joseph Porter 3 , LiChong Xu 1 Show Abstract
1 Surgery, Penn State University, Hershey, Pennsylvania, United States, 2 Bioengineering, Penn State University, Hershey, Pennsylvania, United States, 3 , Juniata College, Huntingdon, Pennsylvania, United States
Protein adsorption to surfaces is a critical event in the interaction of blood with implanted biomaterials, as adsorption/activation of proteins mediates biological responses such as platelet adhesion and thrombus formation. We utilized an array of atomic force microscopy techniques to characterize protein interactions with surfaces, the dynamic structural changes associated with protein/surface interactions and changes in protein activity near the single molecule scale. We find good correlation between molecular scale results and macroscale platelet adhesion as well as differences associated with surface properties of the materials, yielding insights into the mechanisms of biomaterial-induced thrombosis.Fibrinogen is the primary mediator of blood platelet adhesion to materials and as such is a facilitator of biomaterial-induced thrombosis. We previously used AFM to examine structural changes and interaction energies for fibrinogen on surfaces and quantitative analysis of shows fibrinogen undergoes different conformational changes after adsorption to surfaces with different wettability. These interactions are consistent with a two-step spreading process. The results provided new insights into the fundamental interactions between proteins and surfaces and the effects of surface wettability on protein structure/function.Under physiologic conditions, proteins adsorb to surfaces from complicated milieu. We use immuno-modified AFM probes to identify proteins following adsorption from multicomponent solutions. Fibrinogen was recognized on surfaces by polyclonal antibodies that identify the protein and by monoclonal antibodies that recognize exposure of the platelet-binding region. Immunoassay detection showed that the activity of fibrinogen varied with time after adsorption, consistent with conformational changes visualized by AFM, with activity for purified fibrinogen peaking at ~45 min post adsorption. Addition of other proteins to the solution modifies the time to peak activity at towards earlier time points. Macroscale platelet adhesion measurements correlated well with the molecular level functional activity measurements made by AFM. When these measurements were done across a variety of surfaces they showed similar trends but with peak activity shifting in time depending on the properties of the surface. When these data are compiled together and platelet adhesion is compared for similar levels of fibrinogen activity, results show that similar levels of platelet adhesion are seen across all materials we have studied, with the exception of polyurethane biomaterials having a nanoscale chemically heterogeneous surface chemistry. Taken together, our results show that molecular scale structure and function measurements by AFM yield important information on the biological activity of adsorbed proteins and offer new opportunities to understand biological responses to materials.
5:00 PM - U2.7
Scanning Probe Microscopy to Study Electrostatic Interactions of Amyloid Peptides With Model Surfaces and Lipid Membranes.
Brad Moores 1 , Francis Hane 2 , Elizabeth Drolle 2 , Zoya Leonenko 1 2 Show Abstract
1 Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada, 2 Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
Many proteins are known to actively interact with both biologically relevant, as well as inorganic and synthetic surfaces that are widely used in nano- and bio-technology as biosensing platforms and in tissue engineering. Amyloid fibrils are insoluble protein aggregates in beta-sheet conformation that are implicated in at least 20 diseases for which no cure is currently available. The molecular mechanism of fibril formation, as well as the mechanism of fibril clusters interacting with lipid membrane surfaces is currently unknown. The lipid membrane surface has a complex biochemical composition and is also electrostatically non-homogeneous. Currently, the experimental data available for amyloid fibril formation both on lipid and artificial surfaces is limited. The goal of our study is to investigate how the physical properties of the surfaces affect binding of amyloid peptides and affect the fibril formation. We seek to elucidate the effect of electrostatic interactions of amyloid peptides with surfaces using Atomic Force Microscopy (AFM) and Kelvin probe force microscopy (KPFM). I will present a study of Amyloid beta (1-42) fibril formation on model surfaces, which are uniformly charged or possess periodicity of charges and hydrophobic functionality based on thiol self-assembly, as well as on model lipid membrane of various composition. Effect of membrane composition, surface charge, and presence of cholesterol will be discussed.
5:15 PM - U2.8
Local Measurements of Phase Transitions in Bacteriorhodopsin Membrane.
Maxim Nikiforov 1 , Sophia Hohlbauch 2 , William King 3 , S. Antoraz Contera 4 , K. Voitchovsky 5 , Stephen Jesse 1 , Sergei Kalinin 1 , Roger Proksch 2 Show Abstract
1 CNMS, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, 2 , Asylum Research Co., Santa Barbara, California, United States, 3 , University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 4 , University of Oxford, Oxford United Kingdom, 5 , MIT, Boston, Massachusetts, United States
Phase transitions play an important role in biology. Specifically the thermodynamic stability of internal membrane proteins is an important issue because of the difference in biological function for different phases of the proteins. These transitions are associated with the large changes in mechanical properties of the samples. We developed the technique for the measurements of the temperature dependence of the mechanical properties with high spatial resolution. This technique is based on the measurements of the contact stiffness of the atomic force microscopy tip – sample system as a function of temperature by tracking the resonance frequency as it changes. Using this technique, dubbed Dual AC Resonance Tracking (DART) we were able to probe mechanical properties as a function of temperature in the sub-zeptoliter volume. We successfully applied this new technique to the investigate phase transitions in purple membranes. The so-called purple membranes (PM) constitute a large proportion (up to 50%) of the surface area of Halobacterium halobium. PM are composed of only one type of protein, bacteriorhodopsin (bR), and lipids assembled together into an ordered two-dimensional lattice. Phase transitions in BR are the topic which is intensively debated in the last decade. Phase transitions were observed in the wide temperature range from 25°C to 103°C. While there is no consensus about the origin of these transitions, it was found that transitions above 80°C are irreversible, while ones below 80°C are reversible. The transition around 100°C is often attributed to denaturation of components in BR membrane. Various hypotheses for the origin of the transition at lower temperature are proposed:1.Slight change in the chromophore orientation with an alteration of the exitonic interaction.2.Change of the environment of the chromophore within protein.3.Molecular rearrangement of the membrane leading to a less compact protein – lipid packing.The parameters of thermal denaturation of bR, such as the denaturation temperature, enthalpy etc, are good evidences in support either of the hypothesis for the transition in purple membrane.We measured softening temperature of the bR membranes deposited on mica substrate using DART. It was found that extracellular and intracellular membranes have similar softening temperatures. The tip – sample contact area had 20 nm radius meaning that the response from less than 100 molecules was measured. Results of the local heating of bR membrane with heated tip are compared with results obtained after macroscopic heating of the membrane.A portion of this research at Oak Ridge National Laboratory’s Center for Nanophase Materials Sciences was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. A portion of this research was sponsored by Asylum Research Co.
5:30 PM - U2.9
Single Molecule AFM Force Spectroscopy and Steered Molecular Dynamics Simulation of Contactin4 Protein.
Janusz Strzelecki 1 , Karolina Mikulska 1 , Malgorzata Lekka 2 , Andrzej Kulik 3 , Aleksander Balter 1 , Wieslaw Nowak 1 Show Abstract
1 Physics, Astronomy and Applied Informatics, Nicolaus Copernicus University, Torun Poland, 2 Institute of Nuclear Physics, Polish Academy of Sciences, Krakow Poland, 3 Institute of Physics of Complex Matter, Ecole Polytechnique Federale de Lausanne, Lausanne Switzerland
Contactin4 (CNTN4, Big2, 113.454 kDa) is a modular protein, belonging to immunoglobulin superfamily of neuronal cell adhesion proteins (NCAM). It consists of four FnIII and six IgC2 (augmented with disulfide bonds) domains. The protein was identified as being responsible for maintaining and formation of neural circuits in cerebellum and hippocapus. The human CNTN4 (locus 3p26.2 - 3p26.3) was identified as a candidate gene responsible for 3p deletion syndrome, rare contiguous-gene disorder. Mutations affecting CNTN4 function may be also relevant to autism spectrum disorder (ASD) pathogenesis. Previous AFM force spectroscopy experiments have shown that mechanosensitive Fn and Ig type domains are important for regulating cell-cell and cell-extra cellular matrix interactions. Thus understanding nanoscale mechanical properties of Contactin4 has a great medical significance, aiming at diagnosis in a truly nanomedical fashion – one molecule at a time.We used a home made single molecule AFM force spectroscopy setup and steered molecular dynamics (SMD) simulations of single FnIII and IgC2 domains to determine a mechanical behavior of CNTN4 during its unfolding. Saw-toothed force curves, typical for modular proteins were observed, showing at most four unfolding peaks. The analysis of force spectra performed within Worm-Like Chain (WLC) model of polymer elasticity showed the presence of three unfolding lengths: 36.8±1.5 nm, 24,5±0.2 nm and 19.4±1.6 nm. With help of SMD we identified first two cases as resulting from unfolding of respectively native and intermediate states of FnIII. Third case was interpreted as a partial unfolding of IgC2 domain, limited by a disulfide bond. Many force curves have shown repetitive, small (5nm) plateaus bearing some similarities to a hump reported previously during unfolding of titin molecules. A decrease of force in the SMD force extension spectrum, resulting from breaking of hydrogen bonds between G and F strands of FnIII domains provides a possible explanation for this behavior.Mechanical properties of Contactin4 protein suggest, that FnIII serve as a mechanical buffer, which helps to maintain bonds between cells. On the other hand the disulfide bound IgC2 domains unfold only partially. It suggests, that the amount of work necessary to unfold and eventually break the cell-cell bond may be regulated by maintenance or removal of disulfide bonds. SMD calculations for the full CNTN4 model, as well as experiments involving breaking the disulfide bonds are in progress.
5:45 PM - U2.10
A Novel Trimer Packing Motif in the Two-dimensional Nanocrystals of the Drug Molecule Carbamazepine on Au(111) and Cu(111) Substrates.
Erin Iski 1 , Ashleigh Baber 1 , Heather Tierney 1 , April Jewell 1 , Andrew Urquhart 2 , Alastair Florence 2 , Blair Johnston 2 , E. Charles Sykes 1 Show Abstract
1 Chemistry, Tufts University, Medford, Massachusetts, United States, 2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow , Scotland, United Kingdom
Non-covalent intermolecular interactions are of significant interest in understanding the properties and structure of pharmaceuticals. Their influence ranges from solid-state structure, directing 3D packing in polymorphs for example, to drug-drug, drug-excipient and drug-materials interactions during formulation and manufacture. Surface characterization is also of interest in detailing the potential impact that drug-surface interactions may have on the outcome of crystallization processes. Carbamazepine (CBZ) is a dibenzazepine drug widely used in the treatment of epilepsy and, although it has been heavily studied in the solid-state, the intermolecular packing interactions on a 2D scale have not received significant attention. We present results from an ultra-high vacuum scanning tunneling microscopy (UHV STM) study of 2D packing arrangements of CBZ on both Au(111) and Cu(111) surfaces at 78 K. A range of surface coverages and annealing temperatures were studied. On both of the surfaces, the molecules assembled as hydrogen bonded trimers, a packing arrangement that has not previously been observed in 3D structures of this compound. The investigation also revealed that the molecule has a substantially different packing density on the two surfaces with the molecule packed more tightly on the Cu surface. The observations raise the possibility of using molecular packing templates, formed by manipulating substrate-molecule interactions on different substrate surfaces, to template or control polymorphism. Theoretical modeling was also used to examine the packing structures and hydrogen bonding possibilities.
U3: Poster Session: SPM in NanoBioScience
Tuesday PM, April 06, 2010
Exhibition Hall (Moscone West)
6:00 PM - U3.1
Nanoscale Surface Characteristics of Listeria Monocytogenes in Response to Various pH Conditions of Growth.
Bong-Jae Park 1 , Nehal Abu-Lail 1 Show Abstract
1 Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington, United States
Listeria monocytogenes is a facultative intracellular Gram-positive bacterium characterized by strong resiliency to extreme environmental conditions. The in-vivo adherence abilities and further infection capacities of this microbe are significantly affected by environmental pH conditions. Differences in environmental pH can alter the composition of the surface biopolymers of L. monocytogenes leading to altered adhesion and infection capacities. To shed the light on how L. monocytogenes adapt to changes in pH, atomic force microscopy was used to quantify the nanoscale adhesion between L. monocytogenes grown at various pH conditions (pH 5, 6, 7, 8, and 9) and a model surface of silicon nitride. In addition, AFM was used to investigate differences in the nano-mechanical properties of L. monocytogenes cells grown at various pH conditions. Finally, the AFM approach data was used to estimate the thickness and the grafting density of the bacterial surface biopolymer brush as a function of growth media pH. Our results indicate that the specific growth rate constant (μ), carbohydrate to protein ratio (C/P), adhesion force (Fad), Young’s moduli, and biopolymer grafting density (Γ) were maximum when L. monocytogenes cells were grown in a growth media adjusted to a pH value of 7 and were minimum when cells were grown in a growth media adjusted to a pH value of 9. Our results also indicate that the nanoscale adhesion forces (Fad) are directly correlated with the L. monocytogenes specific growth rate, Young’s moduli of cells, and L. monocytogenes biopolymer grafting densities. The correlations can be described as: μ(h-1) = 1.034 * Fad (nN) + 0.093, R2 = 0.847, E (kPa) = 298.757 * Fad (nN) – 55.736, R2 = 0.914, Γ (#/m2) = 2 E16 Fad (nN) + 1 E15, R2 = 0.887
6:00 PM - U3.11
Characterization of Kraft Pulp Fiber Surfaces Using Higher Harmonic Atomic Force Microscopy.
Franz J. Schmied 1 4 , Christian Teichert 1 4 , Lisbeth Kappel 2 4 , Ulrich Hirn 2 4 , Robert Schennach 3 4 Show Abstract
1 , Institute of Physics, University of Leoben, Leoben Austria, 4 , CD-Laboratory for Surface Chemical and Physical Fundamentals of Paper Strength, Graz Austria, 2 , Institute for Paper, Pulp and Fiber Technology, Graz University of Technology, Graz Austria, 3 , Institute of Solid State Physics, Graz University of Technology, Graz Austria
Paper is made up of wood fibers. These natural fibers consist of three main constituents: cellulose, hemicellulose, and lignin. The nano- and microstructure of the fibers, as well as the influence of this structure on the fiber-fiber bond strength are not yet completely understood. To achieve increase in paper strength, a deeper insight into this interrelation is desirable, because the structure has – besides the chemical composition – the most important influence on the mechanical properties. Especially, a closer look on the influence of the individual bonding mechanisms  is desirable to explore the dominating ones. Atomic force microscopy (AFM) seems to be a suitable method to analyze such structural property relation on the nanometer scale.Here, we applied AFM in tapping mode using phase imaging with higher harmonics to study the structure of pulp fiber surfaces and cross-sections. It was possible to visualize the cellulosic microfibrils and their arrangement as well as precipitated lignin on the fiber surface. Additionally, we present investigations of fiber cross-sections showing the individual fiber walls. The results show that AFM is a powerful tool to analyze kraft pulp fiber surfaces as well as fiber cross-sections down to the nanometer scale. Lindström et al., 13th Fundamental Research Symposium (2005), pp 457-562Supported by the Christian Doppler Research Society, Austria and Mondi.
6:00 PM - U3.12
Synchrotron X-ray Tomography and Material Properties of Curly Hair from African Descent.
Guive Balooch 1 , Crystal Porter 1 , Harold Bryant 1 Show Abstract
1 , L'Oreal USA, Chicago, Illinois, United States
Introduction: Hair breakage is one of the most common consumer concerns and although not well understood, the local mechanical properties and composition of hair fibers are critical determinants of how hair fibers fracture under macroscopic conditions. To gain a collective understanding of the localized properties of hair, we employed the novel high resolution techniques of synchrotron XTM, AFM-based nanoindentation, and Elastic Modulus Mapping to asses the material properties and structural modifications of untreated (virgin) fibers that were subsequently treated with chemical hair straighteners (NaOH), surface enhancing molecules, and common cosmetic treatments. Materials and Methods: Synchrotron XTM was used to assess the energy absorption of n=5 hair fibers per group and were performed at the Advanced Light Source (ALS) on Beamline (8-3-2) at the Lawrence Berkeley National Laboratory. This was done by obtaining two-dimensional radiographs as the specimens were rotated through 180 degrees in 0.5 degree increments, with a 750 nm pixel resolution. For relaxer treatment, virgin hair fibers were imaged first, then subsequently relaxed with 2.5% NaOH solution, and imaged again to investigate differences. Results: XTM revealed a significant reduction in fiber energy absorption, seen in the cuticle (45% decrease, p<0.001) and cortex (38% decrease, p<0.01) regions, as a result of NaOH treatment. The energy absorption can eventually be correlated to localized protein concentration, as shown in numerous synchrotron tomography studies of biological tissues. Furthermore, substantial morphological differences as well as surface cracks, voids, and deformations were detected when virgin fibers were treated with NaOH. These results were accompanied by Elastic Modulus Mapping data showing a decrease in elastic modulus in both the cuticle (33% decrease, p<0.01) and cortex (28% decrease, p<0.01) regions of treated hair fibers. When fibers were treated with strengthening molecules, a significant increase in energy absorption was observed compared to virgin fibers. Surprisingly, 3-dimensional XTM reconstructions revealed possible penetration of the strengthening molecules into the cortex of the hair fiber. Conclusion: This is the first study to show the potential of obtaining information on local fractography and energy absorption of hair, using synchrotron x-ray tomography, to better understand the effect of conventional cosmetic treatments. Ultimately, this will lead to a better understanding of how to design new treatments for improving the properties and addressing concerns of ethnic hair.
6:00 PM - U3.13
WITHDRAWN 3/9/10 A New Approach for Measuring the Contact Angle of Nanoparticles at Liquid Interfaces.
Vesselin Paunov 1 , Simeon Stoyanov 2 , Luben Arnaudov 2 , Martien Cohen-Stuart 3 Show Abstract
1 Department of Chemistry, University of Hull, Hull, North Humberside, United Kingdom, 2 Food Structural Design / UFHRI, Unilever Research, Vlaardingen Netherlands, 3 Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Wageningen Netherlands
We have developed a novel generic method for determining of the three-phase contact angle of nanoparticles adsorbed at liquid surfaces. The method includes nano-imprinting of the particle monolayer at the liquid surface with polydimethylsiloxane (PDMS) based on the Gel Trapping Technique (GTT) followed by Atomic Force Microscopy (AFM) imaging of the produced PDMS replicas. The method works for nanoparticles at both air-water and oil-water interfaces and is based on spreading of a sample of nanoparticles at the liquid interface followed by gelling of the aqueous sub-phase with a non-adsorbing polysaccharide. The top phase is replaced by curable PDMS which after its polymerization is peeled off the gel and scanned with AFM. The average contact angle is estimated from the average height of particle protrusion from the PDMS surface and the average radius of same nanoparticles sample measured by AFM in a separate experiment. The novel nano-GTT (GTT+AFM) method allows a significant reduction of the lower particle size limit compared to the SEM imaging used in the original method. We have applied this method to latex nanoparticles, ranging from 37 nm in radius to 120 nm in radius, adsorbed at the air-water and n-decane-water interface. The method is expected to find application as a routine technique for measuring the contact angle of nanoparticles samples adsorbed at a range of liquid surfaces, relevant to a number of food, cosmetic and pharmaceutical formulations.
6:00 PM - U3.14
Kelvin Probe Force Microscopy (KPFM) Measurements of NH3 Sensitive Organic Field Effect Transistors (OFETs) During Operation.
Alexander Bluemel 1 2 , Peter Pacher 2 , Simon Ausserlechner 2 , Harald Plank 3 , Andreas Klug 1 2 , Egbert Zojer 2 , Emil J.W. List 1 2 Show Abstract
1 , NanoTecCenter Weiz Forschungsgesellschaft mbH, Weiz Austria, 2 Institute of Solid State Physics, Graz University of Technology, Graz Austria, 3 Institute for Electron Microscopy, Graz University of Technology, Graz Austria
Organic Field Effect Transistors (OFETs) have been in the focus of research since the late 1980’s. In recent years several works report on Kelvin Probe Force Microscopy (KPFM) measurements on these devices during operation, revealing insight into material properties, intrinsic and fabrication related problems, electronic properties during operation and possible degradation mechanisms. Additionally, also several sensing applications were reported, where a sensing material was introduced e.g. in the active, semiconducting layer. Basically KPFM is an Atomic Force Microscopy (AFM)-based technique and it is used in many fields of application for its capability to reveal the lateral variations of the surface potential on a flat sample.In this contribution we present the results of representative KPFM experiments on NH3 sensitive OFETs, with poly(3-hexythiophene-2,5-diyl) as the active layer and a reactive Self Assembling Monolayer (SAM) as the sensing layer on a Si/SiO2 substrate. In particular, source/drain potential profiles (potential drop across the OFET channel) were measured before and after exposure to NH3 for different operation regimes, i.e. for various source/drain and gate potentials. From electrical characterization presented in another work, a shift of the threshold voltage from ~+70V (before exposure to NH3, due to doping at the interface) to ~0V (after exposure, due to dedoping at the interface after the chemical reaction) could be identified. In this contribution we show that these effects can be correlated to a change in the corresponding source/drain potential profiles across the OFET channel. Additionally, reference devices were characterized (P3HT-OFETs without SAM), showing a threshold voltage of ~0V and potential profiles similar to the NH3-exposed sensor OFET. Thus, with respect to the reference device, the unexposed sensor OFET shows a significant greater threshold voltage and a significant different potential profile, whereas after NH3 exposure both the threshold voltage and the potential profile is similar.
6:00 PM - U3.15
Tip Induced Artifacts in NSOM Characterization of Optical Transmission Through Subwavelength Apertures in Metal Films.
J. Yarbrough 1 , B. Drendel 2 , T. Furtak 2 , P. Flammer 2 , R. Hollingsworth 3 , C. Durfee 2 , R. Collins 2 Show Abstract
1 , NREL, Golden, Colorado, United States, 2 Physics, Colorado School of Mines, Golden, Colorado, United States, 3 , ITN Energy Systems, Inc., Littleton, Colorado, United States
We have explored the use of near-field scanning optical microscopy (NSOM) to image plasmon enhanced optical emission from subwavelength apertures in metal films using both metalized and bare tapered fiber tips. Comparison of measurements with finite element modeling shows that the tip itself can significantly modify the optical field complicating interpretation of the measurements.The plasmonic structures used in this study consisted of an opaque gold film on glass with a single sub-wavelength linear aperture in the film surrounded by two linear gratings. This structure has been shown to transmit many times as much light as a bare aperture due to grating coupling of the incident light into plasmonic modes and resonant cavity effects. The structure shapes the output light into distinct beams due to diffraction from the gratings.Linearly polarized light from a tunable Ti:sapphire laser was incident on the gratings and aperture from the glass side. Collection mode measurements were performed both in the near field by scanning the surface with the tip in feedback, and into the far-field by scanning in the plane perpendicular to the surface. In this way, a 2-D picture of the field intensity moving away from the structure surface was obtained for distances up to 100 microns. Light collection measurements used either tapered bare fiber tips fabricated by heating and pulling the fiber, or commercially available etched metalized tips.2-D finite element simulations of the optical fields (Maxwell’s equations) for the plasmonic structures were performed. Simulations both with and without different types of 2-D analog tips, were used to determine how much the presence of a tip perturbs the fields, and how the tip field collection behaves for both metalized and unmetalized tips. Both experimental results and model simulation show that the presence of the tip perturbs the optical field. The resulting image of the field can differ significantly from the actual field pattern in the absence of the tip. This is particularly true for unmetallized tips, which introduce extra structure into the measured field pattern. Model results assist in understanding why the extra structure arises and how to minimize the effect. Support of the Air Force Office of Scientific Research and National Science Foundation is gratefully acknowledged.
6:00 PM - U3.16
Nanomechanics of Ankyrin Repeats Probed by Single Molecule Atomic Force Spectroscopy.
Whasil Lee 1 2 , Piotr Marszalek 1 2 Show Abstract
1 Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina, United States, 2 Center for Biologically Inspired Materials and Materials Systems, Duke University, Durham, North Carolina, United States
Ankyrin (ANK) repeats, identified in thousands of proteins, are composed of pairs of antiparallel alpha-helices that stack on top of each other and form super-helical spiral domains with suggestive spring-like properties, whose primary function is to mediate specific protein-protein interactions. For example, ankyrin-R links the anion exchanger in the erythrocyte membrane to the membrane skeleton and contains 24 ANK repeats that form a spiral domain. Ankyrin-R stabilizes the erythrocyte membrane and mutations in ANK repeats are documented in hereditary spherocytosis, the life-threatening human anemia, in which red blood cells become spherical and very fragile. However, nothing is presently known about molecular mechanisms underlying the nanomechanical properties of wild type ANK repeats and their mutants. Because ankyrins are only ~10nm in length, the measurements of their elasticity are challenging and require the use of nanotechnology tools such as Atomic Force Microscopy which can manipulate single molecules under nearly in vivo conditions. In this study we exploit single-molecule atomic force spectroscopy to examine the mechanical and folding properties of ANK repeats from ankyrin-R and a model ankyrin repeat protein composed of identical, so called consensus ANK repeats. To ease identification of single molecule recordings we engineered protein hybrids in which eight consensus ANK repeats are flanked by six I27 domains of titin, the best mechanically characterized protein that serves as a mechanical reference. Force elasticity profiles obtained on consensus ANK repeats reveal that these repeats unfold one by one at forces of 20-40pN, producing regular and characteristic saw-tooth patterns of unfolding force peaks. Remarkably, upon relaxation of the stretching force these repeats refold quickly and forcefully, generating strong refolding forces, captured by AFM. ANK repeats from wild type ankyrin-R display similar elastic and unfolding properties and also generate strong refolding forces allowing the molecule to recover its structure under a significant tension. However, unfolding and refolding saw-tooth patterns of ankyrin-R repeats are less regular as compared to consensus ANK repeats. These observations are consistent with the fact that the sequence of ankyrin-R repeats varies significantly between the repeats and, unlike consensus ANK repeats, there may be as yet unidentified hierarchy among mechanical stabilities of ankyrin-R repeats. Importantly, we note that the unique ability of ANK repeats to recover their structure from highly extended conformations, with stretch ratios exceeding 1000% while generating significant refolding forces, may find interesting applications in designing highly resilient bio-inspired springy polymers and materials. Future studies of ANK repeats will focus on identifying the molecular mechanisms of these unusual properties and on examining how ANK repeat mutations affect their elasticity and folding.
6:00 PM - U3.17
Discoidin Domain Receptor 1 Recognizes a Morphological State of Collagen Type 1.
Michelle Nauerth 1 2 , Angela Blissett 2 , Gunjan Agarwal 1 2 3 Show Abstract
1 Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, United States, 2 Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, United States, 3 Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States
Discoidin Domain Receptors (DDR1 and DDR2) are widely expressed receptor tyrosine kinases found in mammalian systems. DDRs bind to and become phosphorylated by triple-helical collagen(s)-the major component of the extracellular matrix (ECM). The fibrillar states of collagen define the mechanical properties of the ECM and govern cell-matrix interactions. The interaction of the DDRs with collagen(s), and their functional implications are not well understood. In this study, we investigated if DDR1 binding and collagen-induced tyrosine phosphorylation is dependent on a specific fibrillar morphology of collagen type 1. Three different morphological forms of collagen type 1: monomeric (M), semi-polymeric (SP) and fibrillar (F) collagen were utilized in our biophysical and cell-based assays. The morphology of the different collagen forms was characterized using nanoscale Atomic Force Microscopy (AFM) imaging under ambient air conditions. Using these characterized collagen morphologies; a biochemical approach such as Western blotting was utilized to determine that DDR1 becomes phosphorylated preferentially by the M form of collagen as compared to F and/or SP forms. However, where DDR1 binds to collagen, as well as its oligomerization state as it binds to the M collagen cannot be ascertained using standard biochemical approaches. Nanoscale imaging is essential in order to elucidate this interaction. We therefore employed single molecule AFM imaging to demonstrate that the DDR1 extracellular domain (ECD)-the region of the protein necessary and sufficient for collagen binding; binds to overlapping monomeric collagen molecules. Quantitative analysis of the AFM images enabled us to ascertain the preferred DDR1 binding sites on the collagen triple helix. Topographic height measurements from AFM images revealed that the DDR1 ECD undergoes oligomerization upon binding to collagen. Furthermore, by using Transmission Electron Microscopy (TEM) and immunogold labeling, we demonstrate that the DDR1 ECD can bind to collagen during collagen fibrillogenesis. Taken together, our AFM and TEM results elucidate how the DDR1 ECD binds to collagen and affects collagen fiber assembly at the molecular level. Our results provide novel insights into how this recognition of the morphological state of collagen by DDR1, modulates collagen fibrillogenesis and collagen regulation via multiple mechanisms.
6:00 PM - U3.2
Three-dimension Imaging of Fibroblast Cells by Scanning Probe Microscope with Cluster Ion Slicing.
Yun-Wen You 1 , Hsun-Yun Chang 1 , Wei-Chun Lin 1 , Bonnie Yu 1 , Che-Hung Kuo 1 2 , Szu-Hsian Lee 1 2 , Chia-Yi Liu 1 , Wei-Lun Kao 1 2 , Guo-Ji Yen 1 2 , Chi-Ping Liu 1 3 , Jing-Jong Shyue 1 2 Show Abstract
1 , Academia Sinica, Taipei Taiwan, 2 , National Taiwan University, Taipei Taiwan, 3 , National TsingHua University, Taipei Taiwan
For the well studied internal structure and abundant rough endoplasmic reticulum, NIH/3T3 fibroblast cell is selected in this work to develop three-dimensional imaging techniques. For high spatial resolution imaging, scanning probe microscopy (SPM) is often used. However, SPM probe interacts only with the outer-most surface of the cell hence it cannot reveal the structure inside the volume. Recently, it is reported that by sputtering the organic surface with 10 kV, 10 nA C60+ and 0.2 kV, 300 nA Ar+ ion beams concurrently, the specimen can be sliced with unappreciated alteration in the remaining surface. As the result, the structure below the outer-most surface can be examined with SPM. By stacking the 2D SPM images acquired at different slices of the specimen, 3D volume image can be reconstructed. Using this novel imaging technique, the internal structure of fibroblast cell is examined. In addition, a scanning (transmission) electron microscope [S(T)EM] based electron tomography operated at 30 kV is used to generate the 3D volume image of fibroblast cell. Images acquired with these techniques are cross-referenced to demonstrate the reliability of the novel technique.
6:00 PM - U3.3
Protein Nanopatterning: Tools and Applications.
Louise Giam 1 , Zijian Zheng 2 , Weston Daniel 2 , Chad Mirkin* 1 2 Show Abstract
1 Materials Science and Engineering, Northwestern University, Evanston, Illinois, United States, 2 Chemistry, Northwestern University, Evanston, Illinois, United States
The ability to fabricate protein micro- and nanoarrays in a low-cost and high-throughput manner is important and relevant to a wide variety of applications, including drug screening, medical diagnostics, biosensors, and fundamental biological studies. Current protein patterning technology relies on photolithography and inkjet printing to generate features at the micron scale, though nanoscale protein patterns would allow for high-density arrays, which can provide increased detection sensitivity. Moreover, proteins form nanoscale architectures in the body, which control many biological processes ranging from cell adhesion to differentiation. In order to mimic this physiological environment properly, nanopatterning techniques are needed where one can place an array of one or multiple protein structures underneath a single cell to study the interactions and biological responses. We present a novel strategy for inking nanoscale polymer probes with different proteins, which can be transferred to a surface through the technique known as Polymer Pen Lithography (PPL)—a recently developed scanning-probe based printing technique. Using this approach, we have generated sub-100 nm protein structures in a rapid manner (150,000 features per second).
6:00 PM - U3.5
Atomic Force Microscopy Imaging of Biological Samples Under Dry and Liquid Conditions.
Larissa Clark 1 , Michel Goedert 2 Show Abstract
1 General Engineering, Bio-Engineering Concentration, San Jose State University, San Jose, California, United States, 2 Chemical & Materials Engineering, San Jose State University, San Jose, California, United States
The purpose of this study was to image biological samples by atomic force microscopy (AFM) in dry and liquid conditions and to optimize the images by varying the probe type and AFM mode. The samples were chromosomes in the metaphase stage of mitosis from normal male lymphocytes and cervical tumor cells. Chromosomes were chosen due to the potential of materials characterization studies of the self-assembly mechanisms of chromosomes and potential applications of self-assembling, programmable bio-materials in the micro- and nano-packaging industries. While contact mode in liquid increased the resolution, high resolution images were also achieved using a form of dry imaging in intermittent contact mode. Where low stiffness probes are often considered optimal for imaging biological samples, we demonstrate that a high stiffness probe in intermittent contact mode can be used to produce high resolution images with no apparent sample damage. This statement is evidenced by consecutive height measurements that showed a percent difference of nominal statistical significance. Our method shows potential for future studies in biological micro- and nano-materials characterization.
6:00 PM - U3.6
On the Deconvolution of Kelvin Probe Force Microscopy (KPFM) Data.
Alexander Bluemel 1 3 , Harald Plank 2 , Evelin Fisslthaler 2 , Meltem Sezen 2 , Andreas Klug 1 3 , Werner Grogger 2 , Emil J.W. List 1 3 Show Abstract
1 , NanoTecCenter Weiz Forschungsgesellschaft mbH, Weiz Austria, 3 Institute of Solid State Physics, Graz University of Technology, Graz Austria, 2 Institute for Electron Microscopy, Graz University of Technology, Graz Austria
Kelvin Probe Force Microscopy (KPFM) is an Atomic Force Microscopy (AFM)-based technique and it is used in many fields of application for its capability to reveal the lateral variations of the surface potential on a flat sample.However, proper interpretation of the real data is often difficult due to a well-known artefact originating from the long range behavior of electrostatic forces: the measured surface potential is crucially affected by the interaction of the cantilever area with the sample surface, leading to a possible misinterpretation of the experimental data.In order to get a better understanding of the measurement process, both the AFM tip and a rectangular shaped sample surface potential distribution were modelled and the tip-sample interaction was simulated, leading to a calculated KPFM image of this test structure. At the same time rectangular test structures were fabricated and measured by means of KPFM. In particular, spin-cast polyfluorene films were selectively irradiated by an electron beam in a Scanning Electron Microscope (SEM). Although simplified, the calculation is capable of showing the influence of the cantilever in the correct qualitative manner: both the simulated and the measured KPFM images show an asymmetrical broadening along the cantilever’s axis of symmetry instead of a pronounced border between the two areas of different surface potential.However, despite the accordance of calculation and experiment, in everyday lab life the more interesting question is, if it is possible to recalculate the real potential distribution from the measured and convoluted images. In this work a deconvolution was tried on the simulated images, showing that for simple geometries it is possible to reveal the "real" surface potential values in principle: areas of equal potential were assigned to the sample surface in the simulated KPFM image. In practice this information could be gathered by e.g. the AFM height or phase image or additional information on the sample. The original potential values are then calculated over a multiple regression. For experimental data the situation is much more complicated: apart from possible numerical problems, several issues as e.g. possible noise within the image make the deconvolution difficult. However, for simple situations a possible way towards a deconvolution of KPFM data could be shown within this work.
6:00 PM - U3.8
Dynamic Force Spectroscopy: Apply the Band Excitation Method to Explore Kinetics of Polymer Conformation Transition.
Senli Guo 1 , Stephen Jesse 1 3 , Maxim Nikiforov 1 , Sergei Kalinin 1 3 , Akhremitchev Boris 2 Show Abstract
1 Center for Nanophase Materials Sciences, ORNL, Oak Ridge, Tennessee, United States, 3 Materials Science and Technology Division, ORNL, Oak Ridge, Tennessee, United States, 2 Chemistry, Duke University, Durham, North Carolina, United States
The dynamic elastic and dissipative behavior on a single molecular level is studied using band-excitation unfolding spectroscopy. In the band excitation approach the cantilever is excited and the response is recorded over a band of frequencies, simultaneously. The evolution of dynamic response at different stages of the unfolding curve is explored using model dextran system. The evolution of dynamic response with force is comparatively studied using multivariate statistical analysis, direct functional fits, and background subtraction methods. Notably, polymer conformation transition is crucial for many biological processes and polymer material properties. The kinetics of polymer conformation transition has been rarely studied because the high transition rate resulting from the low energy barrier among various conformations is beyond the traditional force detection frequency. Combining the band excitation approach the dynamic force spectroscopy measurements can be conducted at a frequency range close to polymer conformation transition rate. This technique can be extended to characterize conformation transition of biological (protein, DNA) and synthetic block copolymers.This research has been conducted at ORNL’s Center for Nanophase Materials Sciences sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. DOE.
Alexei Gruverman University of Nebraska-Lincoln
Igor Sokolov Clarkson University
Zoya Leonenko University of Waterloo
Masamichi Fujihira Tokyo Institute of Technology
U4: Electronic Behavior of Inorganic, Molecular and Biological Systems
Wednesday AM, April 07, 2010
Room 3000 (Moscone West)
9:30 AM - **U4.1
Multifrequency Atomic Force Microscopy.
Roger Proksch 1 Show Abstract
1 , Asylum Research, Santa Barbara, California, United States
One ongoing “holy grail” quest of AFM has been compositional imaging where materials differences are mapped out with the same nanometer resolution as topographic images. There are many forces acting between an AFM tip and a sample, long ranged van der Waals, electrostatic and magnetic forces, short ranged forces stemming from the elasticity of the tip and sample, and dissipative forces associated with adhesion, plasticity, phonon generation, and eddy currents, to name a few. Many if not all of these interactions carry compositional information about the sample.Multiple frequency techniques, where the cantilever motion is measured (and sometimes driven) at multiple resonances or at multiple frequencies around a single resonance have become an active research topic, in part because of their ability to differentiate material properties from the topography of the sample. In the following, we will review some of the emerging techniques and results using AFM cantilevers at multiple frequencies. These will include bimodal techniques  where multiple resonances are excited and the cantilever is operated in a primarily non- or intermittent-contact mode, showing compositional contrast,[2-3] separation of long and short-ranged forces [4-5] and very high resolution, especially of soft, biological materials. The “machinery” developed for bimodal techniques has found other uses in measurements where the cantilever is resonated at two or more frequencies near a single resonance.  One such technique, dubbed Dual AC Resonance Tracking (DART) allows the contact resonance frequency, dissipation, drive phase and amplitude to be extracted from a single measurement. This technique has been applied to piezoresponse force microscopy (PFM), Atomic Force Acoustic Microscopy (AFAM) and Ultrasonic Force Microscopy (UFM) contact resonance measurements and to high resolution localized melting transition measurements using heated cantilevers down to the single molecule level.1T. R. Rodriguez and R. Garcia, "Compositional mapping of surfaces in atomic force microscopy by excitation of the second normal mode of the microcantilever," Applied Physics Letters 84 (3), 449-451 (2004).2R. Proksch, "Multifrequency, repulsive-mode amplitude-modulated atomic force microscopy," Applied Physics Letters 89 (11), 3 (2006).3N. F. Martinez et al., "Enhanced compositional sensitivity in atomic force microscopy by the excitation of the first two flexural modes," Applied Physics Letters 89 (15) (2006).4J. W. Li, J. P. Cleveland, and R. Proksch, "Bimodal magnetic force microscopy: Separation of short and long range forces," Applied Physics Letters 94 (16) (2009).5B. J. Rodriguez et al., "Intermittent contact mode piezoresponse force microscopy in a liquid environment," Nanotechnology 20 (19) (2009).6B. J. Rodriguez et al., "Dual-frequency resonance-tracking atomic force microscopy," Nanotechnology 18 (47) (2007).
10:00 AM - U4.2
Nonlinear Interaction Imaging and Spectroscopy in Scanning Probe Microscopy.
Stephen Jesse 1 , Sergie Kalinin 1 Show Abstract
1 , Oak Ridge National Lab, Oak Ridge TN, Tennessee, United States
All information about material properties acquired through scanning probe microscopy is derived from the measurements of interactions between the tip and the sample. Nearly every form of scanning probe technique developed to date is based on measuring and recording only the linear aspects of this interaction and disregarding the nonlinear components. Although, much can be derived from looking at only the linear interactions, there is perhaps more important information hidden within the nonlinear ones such as local field gradients, indentation forces, hysteresis associated with anelastic and ferroic effects etc.. Of particular value are the short range forces that are generated when the tip is closest to the surface, that contain information on specific interactions and binding. We have developed an atomic force microscope control and acquisition scheme that can determine both the linear and non-linear components of tip-surface interaction and thereby extract both the usual metrics of tip motion, such as amplitude, resonance, and dissipation, as well as the force-distance curves for arbitrary tip-surface interactions. This method is based on performing non-linear operations on measured data and including this in least-squares fit to a non-linear model. We will demonstrate this approach for modeled data, and experimental data including force-distance curves, tapping mode, piezo-response force microscopy (contact mode), and magnetic force microscopy (non-contact mode). The methodology developed here can be used as a general protocol applicable to the measurement and analysis of wide variety of oscillating systems.Research at Oak Ridge National Laboratory's Center for Nanophase Materials Sciences was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy.
10:15 AM - U4.3
Novel 3D Piezoresponse Force Microscopy: Decoupling the In-plane and Out-of-plane Tip Motions.
Seungbum Hong 1 , Ramesh Nath 1 3 , Moonkyu Park 4 , Bryan Huey 5 , Kwangsoo No 4 , Orlando Auciello 1 2 , Petford-Long Amanda 1 Show Abstract
1 Materials Science Division, Argonne National Laboratory, Lemont, Illinois, United States, 3 Institute of Functional Materials, University of Puerto Rico, San Juan, Puerto Rico, United States, 4 Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejon Korea (the Republic of), 5 Institute of Materials Science, University of Connecticut, Storrs, Connecticut, United States, 2 Center for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois, United States
We present our recent results that reveal the effects of cantilever buckling, induced by the in-plane (IP) motion of the atomic force microscopy (AFM) tip, on the out-of-plane (OP) polarization signal when imaging polarization domains, using piezoresponse force microscopy (PFM). In order to quantitatively understand the contribution of the cantilever buckling to the OP polarization signal, we studied BaTiO3 (BTO) single crystal and BiFeO3 thin film based nanostructures in order to compare weakly and strongly coupled IP-OP PFM signals. For BTO single crystal, which exhibit weakly coupled IP-OP signals, we found no correlation between IP and OP PFM images and no change in OP signal as a function of rotation angle when rotating the sample in the plane around an axis perpendicular to the surface, whereas for BFO nanostructures, which exhibit a strong IP-OP signal coupling, we observed a strong correlation between the OP signal for the sample oriented in the plane at 0 degree (defined as the direction of motion of the AFM tip) and the IP signal measured when the samples is rotated 90 degrees on the plane with respect to the initial 0 degree direction as defined above. In addition, we found that the IP-OP PFM coupling depends strongly on the laser position with respect to the support end of the cantilever. In the case of BTO with weakly coupled IP-OP signals, the IP PFM amplitude signal deviates from the ideal model probably due to the unstable contact condition, which makes conventional vector PFM a challenging task. Based on our findings, we introduce two novel PFM methods, namely: a) angle-resolved PFM and b) in-situ 3D PFM, which can be used to construct 3D PFM images of polarization domains with better efficiency and reliability than the current methods that do not distinguish the IP-OP coupling effect.
10:30 AM - **U4.4
Functional Recognition Imaging and Artificial Intelligence Methods in Scanning Probe Microscopy.
Sergei Kalinin 1 , Oleg Ovchinnikov 1 , Stephen Jesse 1 Show Abstract
1 , ORNL, Oak Ridge, Tennessee, United States
Scanning probe microscopy (SPM) techniques have become the mainstay of nanoscience and nanotechnology by providing easy-to-use, non-invasive structural imaging and manipulation on the nanometer and atomic scales. The rapid emergence of spectroscopic imaging techniques in which response to local force, bias, or temperature is measured at each spatial location necessitates the development of data interpretation and visualization techniques for 3- or higher dimensional data sets. In this talk, I will briefly summarize recent advances in applications of neural network based artificial intelligence methods in scanning probe microscopy. This approach utilizes statistical analysis of complex SPM responses to identify the target behavior, reminiscent of associative thinking in the human brain and obviating the need for analytical models. As an example of recognition imaging, we demonstrate rapid identification of cellular organisms using difference in electromechanical activity in a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method. As a second example, I demonstrate the use of neural network recognition for analysis of random bond-random field Ising model parameters in ferroelectric capacitors. The future prospects for smart SPMs are discussed. Research at Oak Ridge National Laboratory's Center for Nanophase Materials Sciences was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy.
11:00 AM - U4: Electronic
11:15 AM - U4.5
Nanoscale Electronic Property Study of a Model System and of a Conjugated Polymer Compound by Kelvin Probe Force Microscopy and Scanning Conductive Torsion Mode Microscopy.
Ling Sun 1 , Wang Jianjun 2 , Gerhard Wegner 2 , Hans-Juergen Butt 1 , Elmar Bonaccurso 1 Show Abstract
1 Polymer physics, Max Planck institute for polymer research, Mainz Germany, 2 Solid state chemistry, Max Planck Intitute for polymer research, Mainz Germany
The properties of conjugated polymers are influenced by their nanoscale morphology. These features affect charge injection, transport, recombination, and trapping on a sub-100 nm length scale when the conjugated polymers are incorporated in electronic devices. Electrical atomic force microscopy is an ideal tool to measure simultaneously electronic properties and surface morphology of thin films of conjugated polymers. For instance Kelvin Probe Force Microscopy (KPFM) and Electrostatic Force Microscopy (EFM) enable to directly measure surface potentials, which are used to study mesoscopic charge injection and charge trapping effects in active electronic devices. Conductive-AFM (c-AFM), Scanning Tunneling Microscopy (STM), and the recently presented Scanning Conductive Torsion Mode Microscopy (SCTMM) can probe the heterogeneity of charge transport in thin films of organic semiconductors. Nevertheless all the above mentioned technologies have their own limits in correlating the electronic properties, i.e. surface potential and conductivity, and the morphology of conjugated polymers at the nanoscale. As an example, poly (3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) complex is a mixture of a conductive component that determines the electronic property, and a non-conductive amorphous component that increases the processability of the complex. Information on conductive domains that are completely surrounded by the matrix is not always accessible by c-AFM or SCTMM, unless the conductive domains form a conducting channel (percolation path) through the sample. On the other hand, the surface potential measured by KPFM is a weighted average of all contributions close to the apex of the sensing tip, which decreases the lateral resolution of KPFM to 10-100 nm. Thus when the distance between two adjacent domains is beyond the resolution of KPFM, the measured surface potential turns out to be an average of the two domains, leading to an ambiguous result. Here we present how to overcome the limitations of both techniques by a combined analysis. We demonstrate it first on a model system with controlled nanoscale morphology and known electronic properties. We will later show that we can apply this knowledge to describe the electronic properties of a conjugated polymer compound we synthesized: polypyrrole:polystyrene sulfonate (PPy:PSS).The model we used was a gold/polystyrene (Au/PS) system, with conductive gold nanoparticles (AuNPs) embedded into a non-conductive polystyrene (PS) film. The AuNPs resemble the conductive domains and the PS the non-conductive polymer matrix. The average diameter d of AuNPs is ~30 nm, which is similar in size to the conductive domains in conjugated polymers.2, 17 We controlled the nanoscale morphology by varying two parameters: (i) the distribution of the AuNPs inside the PS film (from individual particles to small to large clusters); (ii) the thickness of the PS layer around and on top of the AuNPs.
11:30 AM - U4.6
Characterization of Electrical Properties of Biological Materials Using Scanning Eddy Current Force Microscopy With Nanometer Resolution.
Vijay Nalladega 1 , Shamachary Sathish 1 , Karolyn Hansen 2 , Douglas Hansen 1 Show Abstract
1 , University of Dayton Research Institute, Dayton, Ohio, United States, 2 Department of Biology, University of Dayton, Dayton, Ohio, United States
Characterization of the electrical properties of biological materials is important in several areas of physiology and biophysics. For example, electrical impedance measurements can be used to identify malignant tumors. Various techniques have been developed to measure and image electrical properties of biological materials on macro scale. Since the invention of AFM, it has been extensively used to characterize the surface morphology of biological materials. One of the biggest advantages of the AFM for biological material characterization is that it can be used to image the biological materials in their own physiological conditions. The AFM has also been modified to image electrical properties of biological materials. In these techniques, a bias voltage is applied between a conducting tip and the sample surface to image the resulting electrical and electromechanical interactions. In some cases, the bias voltage applied may modify the local electrical properties of some biological samples. Moreover, in these methods, the measurements are performed at each location by moving the tip from one discrete location to the next across the sample. Therefore, these techniques are quite time consuming. In this study, we use the recently developed atomic force microscopy based eddy current microscopy (Nalladega et al. Rev. Sci. Instrum. 79, 073705 (2008)) to characterize the electrical properties of biological materials. The methodology is based on the combined principles of eddy currents and traditional AFM. The eddy currents generated in the material are detected and measured using a magnetic tip attached to a flexible cantilever of the AFM. The new methodology is used to characterize the electrical properties of bovine serum albumin and oyster hemocyte cells. The contrast in the images is explained based on the local variations in ionic conductivity of the samples. The advantages and applications of the technique to biological material characterization are discussed.
11:45 AM - U4.7
Electronic Transport Through Materials-selective Peptides.
Yuhei Hayamizu 1 , Christopher So 1 , Hilal Yazici 1 2 , Marketa Hnilova 1 , Ersin Emre Oren 1 , Candan Tamerler 1 2 , Mehmet Sarikaya 1 Show Abstract
1 Genetically Engineered Materials Science and Engineering Center, Materials Science and Engineering Department, University of Washington, Seattle, Washington, United States, 2 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey
Bioelectronic and bioenergetics devices are based on hybrid systems consisting of biological materials, such as proteins, enzymes, and DNA, and inorganic materials as electrodes. In these devices, the interfaces between these biomacromolecules and inorganic substrate materials are critical for the transduction of electronic signals from molecules to electronic components and vice versa. The electronic transport through proteins has been extensively investigated in energy conversion processes, for example, in photosynthesis, respiration, and enzymatic processes. Conventionally, synthetic molecular systems, such as silanes or thiols, are widely used as linkers to immobilize biomacromolecules at solid interfaces. As a novel alternative to traditional linkers, a new class of peptides, GEPIs, genetically engineered peptides for inorganics, could be used because of their materials-selectivity and binding properties. These peptides are genetically selected using biocombinatorial methods, e.g., phage display and cell surface display peptide libraries, and their binding, materials selectivity, and assembly characteristics are quantitatively investigated using surface plasmon resonance spectroscopy and atomic force microscopy (AFM). GEPIs have biocompatibility, which is crucial for the application in biological environment. They enable controlled attachment of nanoentities to targeted domains and as well as assemble ordered molecular array. Using these solid-binding peptides, here we characterize their electronic transport using scanning tunneling microscopy (STM) and AFM. As a typical platform, gold binding peptides (AuBPs) and graphite binding peptides (GrBPs) with specific binding affinity to gold and graphite surfaces, respectively, are used. The AuBPs self-assembled and immobilized on Au (111) surface. Several types of peptides, which have different binding affinity were studied to compare the electronic transport properties. Our STM characterizations on several AuBPs indicate significantly different current vs. voltage (I-V) characteristics through the peptides on the same Au substrate. Although the molecular mechanisms are not known, we attribute these different behaviors of I-V characteristics to the different peptide conformations that originate the differences in amino acid sequences of the peptides. Similarly, GrBPs were assembled on graphite surface and their electron transport properties were investigated. The binding versus transport characteristics were also studied using AuBPs on graphite (111) substrate and GrBPs on Au (111) substrate to quantitatively shed light into molecular recognition and, specifically, binding versus electron conductivity of these dodecapeptides as a first step towards their utility in practical bioelectronics. The research was supported by Genetically Engineered Materials Science and Engineering Center (GEMSEC), an NSF-MRSEC at the UW and an NSF-BioMat grant.
12:00 PM - **U4.8
Bioinspired and Biological Peptide Nanotubes: SPM Nanoscale Characterization, Basic Physics and Applications.
Gil Rosenman 1 Show Abstract
1 Physical Electronics, Tel Aviv University, Tel Aviv Israel
Elementary biological units proteins and peptides have the intrinsic ability to self-assemble into elongated solid nanofibrils-natural biological nanotubes, which give rise to amyloid diseases (Alzheimer, Parkinson, etc). It has been found (Reches and Gazit, Science, 2003) that core recognition motif of Alzheimer's Aβ peptide the diphenylalanine self assemble into well ordered peptide nanotubes (PNT) This lecture will be focused both on recently developed a new preparation process-vapor deposition of biolomolecules for making highly ordered alignments of PNT and describe physical properties studied in these bioinspired self assembled short aromatic PNT by the use of SPM-PFM. The conducted studies show that PNT as well biological amyloid nanotubes possess nanocrystalline structure characterized by quantum confinement and luminescence of exciton origin. This bio-inspired ceramic nanostructural material, having nonsymmetric structure, demonstrates anomalously strong piezoelectric activity (cooperation with Dr. A. Kholkin), pointing to electric polarization directed along the tube axis. High piezoelectric shear coefficient measured by PFM is about 70-100 pm/V and it strongly exceeds the longitudinal one ~7 pm/V for d33. PNT show high intensity second harmonic generation with nonlinear optical coefficient - 20 pm/V (cooperation with Prof. Mishina). We will also report on the development of bottom-up nanotechnology of fabrication of PNT arrays, bundles and templates. These findings permit to propose a new advanced directions both in bio-ferroelectricity and in bio-nanotechnology, based on surprising physical properties at interchange Physics-Biology toward a fundamental research of a novel group of environmentally clean bio-inspired nanostructural peptide nanotube materials and a new generation of piezoelectric, energy storage and photonic nanodevices. References1. L. Adler-Abramovich, D. Aronov, P. Beker, M. Yevnin, L. Buzhansky, G. Rosenman, E. Gazit, Nature Nanotechnology, 18 October, DOI: 10.1038/NNANO.2009.298 (2009) 2. N. Amdursky, M.Molotskii, D.Aronov, L. Adler-Abramovich, E. Gazit, G. Rosenman, Nano Letters, 9, 3111, (2009) 3. N. Amdursky, M. Molotskii, E. Gazit, G. Rosenman, Appl Phys. Letts., 94, 261907(2009) 4. D. Aronov, L. Adler-Abramovich, E. Gazit, G. Rosenman, J. Nanosci. Nanotech., 8:1-8 (2008)
12:30 PM - U4.9
A Novel AFM-MEA Platform for Studying the Real Time Mechano-electrical Behavior of Cardiac Myocytes.
Jose Saenz Cogollo 1 , Mariateresa Tedesco 1 , Sergio Martinoia 1 , Roberto Raiteri 1 Show Abstract
1 Department of Biophysical and Electronic Engineering, Università degli Studi di Genova, Genova, GE, Italy
In the present work we propose a new analytical platform based on the combination of two techniques: Atomic Force Microscopy (AFM) and Micro-Electrode Arrays (MEA) for performing precise-and-local mechanical stimulations on living cells while measuring in situ and in real time changes in their extracellular electrical activity. We applied this combined set-up to mechanically stimulate a single cardiac myocyte (CM) and study the resulting electrophysiological response. Such approach can be helpful in the understanding of the cardiac Mechano-Electrical Feedback (MEF) phenomenon which is involved in the adjustment of heart rate, the initiation of arrhythmias, and the re-setting of disturbed heart rhythm by ‘mechanical’ first aid procedures. Despite all the work done in the exploration of the effects and mechanisms behind the MEF, the process of signal transduction in MEF at the single cell level is not completely understood yet. In order to elucidate the (sub)cellular mechanisms and processes of signal transduction in the cardiac MEF phenomenon, AFM looks particularly promising since it allows the application of controlled low forces, the measurement of the mechanical properties at the point of stimulation, a minimal disruption to the membrane, and the determination of cellular strain distribution during indentation. On the other hand, recording of extracellular field potentials from contracting CM without interfering with cells motility or producing undesirable side effects is possible when the cells are grown on the glass surfaces with integrated electrodes of the MEA technology.We have combined commercial AFM and MEA instrumentation so that the AFM sits onto the MEA connector while both are positioned onto an inverted optical microscope. In this way it is possible to check with accuracy the position of the AFM tip over the MEA. The electrical and mechanical noise in the developed platform is comparable with the noise in the standard configuration of both instruments. In initial experiments we studied monolayer and patterned cultures of CM from rat embryos. First we characterized the mechanical and electrical spontaneous activity of contracting cells. Then we applied controlled pulses of force, peak force in the range 1nN to 65nN and duration in the range 10ms to 100ms, onto the membrane of cells sitting on top of or near to recording electrodes. We could clearly observe electrophysiological responses, especially when applying force pulses greater that 40nN. By changing the delay between the spontaneous electrical spikes and the stimulation, we observed that the waveforms of the evocated activity are in agreement with the possible activation of stretch-activated ion channel and even though more experiments are needed to be conclusive, the variability in the amplitude of the responses, in relation to the spatial position of the stimulation, suggests a strong relation between the mechano-sensitivity and the local membrane-cytoskeleton structure.
12:45 PM - U4.10
Why CA FM-AFM Works Well and How to Image Even More Gently.
Paul Ashby 1 Show Abstract
1 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States
Recently constant amplitude frequency modulation AFM (CA FM-AFM) has become popular for imaging of soft materials. This is a result of the feedback loops involved with CA FM-AFM modulating the effective damping and effective temperature of the cantilever. Q-control amplitude modulation AFM (AM-AFM) has a similar feedback loop for adjusting the effective damping but has not achieved similar quality results. I will present cantilever trajectory simulations which contrast CA FM-AFM with Q-control AM-AFM and explain the physical origin of the superior performance of CA FM-AFM. The results of this investigation suggest that changes in cantilever geometry which reduce the intrinsic damping of the cantilever is the optimal means of reducing tip-sample interaction force while retaining high signal-to-noise ratio.
U5: Biomolecules and Bioassemblies II
Wednesday PM, April 07, 2010
Room 3000 (Moscone West)
2:30 PM - **U5.1
Atomic Force Microscopy Measurements of High Resolution Structure of Bacterial Cell Sacculi.
Ahmed Touhami 1 , Valerio Matias 2 , Anthony Clarke 2 , Manfred Jericho 3 , Terry Beveridge 2 , John Dutcher 1 Show Abstract
1 Department of Physics, University of Guelph, Guelph, Ontario, Canada, 2 Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, 3 Department of Physics and Atmospheric Science, Dalhousie University, Halifax, Nova Scotia, Canada
The cell envelope of Gram-negative bacteria is responsible for several important biological functions: it accommodates the selective transfer of molecules into and out of the cell, and it plays a crucial structural role in maintaining the integrity of the cell wall during growth and division of the cell. The main structural component of the cell envelope is the peptidoglycan sacculus, one of nature’s largest and strongest biopolymers. The intricate nature of its structure and biosynthesis pathway make peptidoglycan one of Nature’s marvels of nanotechnology but also allow it to be the principal target of many antibiotics and one of the main microbial products recognized by the immune system. It is a covalent macromolecular structure of stiff glycan chains that are crosslinked by flexible peptide bridges. Although the peptidoglycan sacculus has been the subject of decades of investigation, its construction and higher-order structure are still debated. Two models have been proposed for the architecture of the peptidoglycan network (orientation of the glycan strands parallel or perpendicular to the cytoplasmic membrane), but it has not been possible to date to determine which model is correct. By collecting atomic force microscopy (AFM) images and force-distance curves on purified intact Escherichia coli K12 sacculi, and exposing the sacculi in situ to an AmiB amidase enzyme, we have obtained strong evidence that the popular planar model in which the glycan strands are arranged parallel to the cytoplasmic membrane is the correct model for the peptidoglycan network in this Gram-negative bacterium.
3:00 PM - U5.2
Towards Physical Methods of Detection of Cancerous Cells.
Igor Sokolov 1 3 4 , Ravi Gaikwad 1 , Maxim Dokukin 1 , Nataliia Guz 1 , Swaminathan Iyer 5 , Craig Woodworth 2 Show Abstract
1 Physics, Clarkson University, Potsdam, New York, United States, 3 Chemical and Biomolecular Science, Clarkson University, Potsdam, New York, United States, 4 Nanoengineering and Biotechnology Laboratories Center (NABLAB), Clarkson University, Potsdam, New York, United States, 5 Chemistry, The University of Western Australia, Crawley , Western Australia, Australia, 2 Biology, Clarkson University, Potsdam, New York, United States
To date, the methods of detection of cancer cells have been mostly based on traditional techniques used in biology, such as visual identification of malignant changes, cell growth analysis, specific ligand-receptor labeling, or genetic tests. Despite being well developed, these methods are either insufficiently accurate or require a lengthy complicated analysis. A search for alternative methods for the detection of cancer cells may be a fruitful approach. Here we describe a different, physical approach to detect cancer cells in vitro. We use atomic force (AFM), laser scanning confocal (LSCM), and electron (both SEM and TEM) microscopies to study the differences in surface properties of normal and cancerous epithelial cervical cells. We demonstrate that AFM is the most convenient, numerically accurate, and fastest method to obtain information about the surface properties of cells. Several methods of detection of cancerous cells derived from the found surface differences are presented.
3:15 PM - U5.3
Investigation of Single-walled Carbon Nanotubes and Antibacterial Activity by Atomic Force Microscopy.
Yuan Chen 1 , Shaobin Liu 1 Show Abstract
1 School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore Singapore
The advent of engineered single-walled carbon nanotubes (SWCNTs) in commercial and industrial applications raised concerns about their potential impacts on human health and environmental safety. Atomic force microscopy (AFM) is an ideal tool for nanoscale analysis of microbial cells, which provides high resolution images of cell membranes. In this study, the interactions between SWCNTs and bacteria (both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Bacillus subtilis) were investigated by AFM. The AFM images of bacteria were obtained after their interactions with SWCNTs over different periods of time. These images show distinct changes in the cell envelope morphology over different exposure periods. SWCNTs are able to cause significant damages on the bacterial membranes, which is responsible for changes in the cell membrane permeability and the leakage of cytoplasm. Furthermore, AFM study also indicated that the death rates of bacteria were strongly correlated with their cell membrane stiffness; soft cells were more vulnerable to SWCNT piercing. Our results support the hypothesis that the antibacterial activity of SWCNTs are caused by the physical puncture of cell membranes.
4:00 PM - **U5.4
Soft Matter Research by Scanning Probe Techniques in Cosmetics.
Gustavo Luengo 1 Show Abstract
1 , L'Oreal, Aulnay sous Bois France
Traditionally the use of Scanning Electron Microscopy and Transmission Electron Microscopy has helped understand the complex structure of cosmetic substrates (hair and skin) and to evaluate the impact of materials of cosmetic interest on its structure. With the advent of Atomic Force Microscopy (AFM) it has become possible to access many physical properties of these “biocomposites” with the advantage of being able to use this technique in ambient conditions or in liquids.We will present a review of different and various applications of AFM in cosmetic research, in particular the methods used to explore the complex structure of skin and hair and its modification by materials (polymers).For example, we have been interested in the friction and adhesion (tribology) properties of hair and upper skin cells (corneocytes) as well as the subtle alterations induced by water and other materials of cosmetic interest. In addition, and in order to localize the contribution of hair and skin cell constituents to the overall macroscopic structure, we will discuss the methods used to bring out their mechanical properties.Finally, physicochemical local information can also be obtained from the use of chemically modified AFM tips. The use of this technique is particularly promising as a way to quantify the chemical groups available at the substrate surface, which are responsible for successful adsorption of a given material onto it. Combination of these versatile AFM techniques is highly interdisciplinary and helps us to advance towards the identification of the properties of the substrates and most importantly to understand the way a specific cosmetic treatment (new active or material) alter them.
4:30 PM - **U5.5
Scanning Probe Microscopy Insights into Supramolecular π-Conjugated Nanostructures for Optoelectronic Applications.
Philippe Leclere 1 2 , Mathieu Surin 1 , Roberto Lazzaroni 1 , Albertus Schenning 2 , Bert Meijer 2 Show Abstract
1 , University of Mons (UMons), Mons Belgium, 2 , Technische Universiteit Eindhoven (TU/e), Eindhoven Netherlands
Well-defined conjugated materials play an important role in the growing field of organic electronics because their precise chemical structure and conjugation length give rise to well-defined properties and facilitate control over their supramolecular organization. The building of nanoscopic and mesoscopic architectures represents a starting point for the construction of (supra)molecular electronics or even circuits, through surface patterning with nanometer-sized objects. It clearly appears that the solid-state properties of organic electronic materials are determined not only by those of individual molecules but also by those of ensembles of molecules. The ability to control the supramolecular architectures is thus essential for optimizing the properties of conjugated materials for their use in technological applications in the field of nanoelectronics. Within the two past decades, many new Scanning Probe Microscopy (SPM) operational modes have been successfully introduced, giving birth to a family of SPM for the high resolution characterization of organic and polymer thin films in terms of their electrical, magnetic, thermal, and optical properties, as well as their chemical composition. All these advances have rendered SPM tools of choice for studying self-assembled organic structures arising from specific supramolecular assembly of (macro)molecules, from intermolecular interactions (such as hydrogen-bonding and/or π-π interactions), or from phase separation in block copolymers or polymer blends. In this work, we report on the observation by atomic force microscopy (AFM) of nanoscale architectures obtained in the solid-state from solutions of conjugated materials, and demonstrate that they can organize onto a surface over lengthscales from nanometers to several microns, forming semiconducting nano-objects by π-stacking processes.We will emphasize some recent contributions of SPM’s to the field of organic electronics, from the morphological studies of novel supramolecular materials to a better understanding of the optoelectronic properties and device performances. Recent works for controlling the nanoscale ordering of π-conjugated molecules, carried out by taking advantage of self-assembly will be described for bio-templated assemblies (using ssDNA) and for nanophase separation in block copolymers. We focus on the use of SPM’s for a better understanding of the relationship between the (nano)morphology and performances of light-emitting devices (e.g. for white-lighting applications), field-effect transistors, and organic photovoltaic diodes for solar cells applications.
5:00 PM - U5.6
Type I Collagen Exists as a Distribution of Nanoscale Morphologies in Teeth, Bones and Tendons.
Joseph Wallace 1 , Qishui Chen 1 , Ming Fang 1 , Blake Erickson 3 , Bradford Orr 2 , Mark Banaszak Holl 1 Show Abstract
1 Chemistry, University of Michigan, Ann Arbor, Michigan, United States, 3 Biophysics, University of Michigan, Ann Arbor, Michigan, United States, 2 Physics, University of Michigan, Ann Arbor, Michigan, United States
Type I collagen forms the structural scaffolding upon which many tissues are built. The current study demonstrates that regardless of cellular origin, anatomical location or mechanical function, Type I collagen-based tissues contain a distribution of nanoscale collagen morphologies as measured using Scanning Probe Microscopy. The presence of these distributions is a new and important observation on the nanoscale ultrastructure of the most abundant protein in animals and may hold powerful information about the ultrastructure of collagen-based tissues. Monitoring how these distributions change may represent a new diagnostic technique for damage and disease.It was hypothesized that AFM could be used to image and quantitatively analyze the morphology of Type I collagen fibrils in fully intact and mineralized teeth and bones as well as in non-mineralized tendons. These tissues were chosen based on differences in cellular origin and function, as well as to compare mineralized tissues with a tissue which lacks mineral under normal physiological conditions. Tissues were harvested from 8 week old male Sv129/CD-1/C57BL/6S mice (UCUCA protocol #09637). Images were acquired from 3 locations in each of 5 teeth, 9 locations in each of 4 bones and several locations in each of 4 tendon samples. A region of interest was chosen along straight segments of individual fibrils, a two dimensional Fast Fourier Transform was performed and the primary peak from the 2D power spectrum was used to determine the value of the D-periodic spacing. Measurements within each sample were pooled to yield the mean fibril spacing for that sample (67.8 nm, 67.3 nm and 67.9 nm for dentin, bone and tendon, respectively). When compared by One Way ANOVA with post-hoc Bonferroni tests, no significant differences were present between any of the groups.A distribution of fibril morphologies existed in each tissue, an interesting and important observation which may hold the key to understanding important aspects of the ultrastructure of many collagen-based tissues. To test for differences in distributions, the Cumulative Distribution Function (CDF) was computed from the measurements in each tissue, and a Kolmogorov-Smirnov test was applied to the CDFs. The distributions from the three tissue types were statistically indistinguishable from one another. The concept of a distribution is often overlooked in measurements of collagen, and the mean value for the D-periodic spacing is reported without explanation. The presence of a distribution was a common feature in all three tissues, indicating that the distribution is a defining nanoscale characteristic of Type I collagen. Distributions were present both in mineralized tissues and a tissue which does not mineralize under normal physiological conditions, verifying that the presence of mineral is not the source of the observed distributions.
5:15 PM - U5.7
Improving AFM of Cells Using CNT Probes.
Jessica Koehne 1 2 , Ramsey Stevens 3 , Tiffany Zink 1 , Zhao Deng 1 , Huan-yuan Chen 1 , I-Chun Weng 1 , Fu-tong Liu 1 , Gang-yu Liu 1 Show Abstract
1 , University of California, Davis, Davis, California, United States, 2 , NASA Ames Research Center, Moffett Field, California, United States, 3 , Carbon Design Innovations, Burlingame, California, United States
While atomic force microscopy (AFM) has become important alternative approach to visualizing membrane morphology of individual cells, many studies have reported the presence of artifacts such as cliffs on the edges of cell. These artifacts shield important structural features such as lamellopodia, filopodia, microvilli and membrane ridges, which are of great importance to resolve cellular signaling processes and activation. High aspect ratio carbon nanotube (CNT) modified AFM probes of varied spring constants and geometries were tested by imaging rat basophilic leukemia (RBL) cells. In all experiments, CNT probes reveal little cliff-like artifacts and enable complete visualization of entire membrane morphology and surface features. The mechanism of cliff-like artifact is revealed based on the AFM probe’s geometry. This work represents the first time that whole cells have been imaged with CNT tips and offers a remedy to the cliff-like artifact generation that has limited the acceptance of AFM in cellular biology.
Alexei Gruverman University of Nebraska-Lincoln
Igor Sokolov Clarkson University
Zoya Leonenko University of Waterloo
Masamichi Fujihira Tokyo Institute of Technology
U6: SPM Instrumentation
Thursday AM, April 08, 2010
Room 3000 (Moscone West)
9:30 AM - **U6.1
Single-path Kelvin Force Microscopy Imaging in Different Environments.
Sergei Magonov 1 Show Abstract
1 , Agilent Technologies, Chandler, Arizona, United States
Applications of Atomic Force Microscopy (AFM) to different materials are benefit from developments of high-resolution imaging, probing of local mechanical and electromagnetic properties. The improvement of the microscope characteristics and expansion of its capabilities to broadband and multi-frequency detection are essential for this progress. Recently we applied single-path Kelvin Force Microscopy (KFM) to analysis of complex materials. The method was implemented with frequency modulation detection of the tip-sample interactions and measurements were performed in the intermittent contact mode1. The value of this approach was verified in studies of semiconductors, metals and self-assembled organic systems2-3. It was noticed that KFM is not affected by screening at high humidity when imaging is performed in the intermittent contact. Therefore the method can be applied in different environments, and studies of polymers with hydrophilic components will be presented below. AFM imaging of heterogeneous materials in different vapors is very useful. Annealing of block copolymer in vapor of common solvent can be monitored with AFM in-situ. Studies of complex polymer materials in vapors of selective solvents, which induce swelling of individual components, might help recognizing their morphology. We have performed KFM of several polymers in different vapors. The surface potential maps, which were recorded at high humidity, helped distinguishing the hydrophilic components and their distribution. Blends of polystyrene (PS) and poly(methyl methacrylate) (PMMA) and block copolymer (PS-b-PMMA) were examined as the model systems. A microphase separation of PS-b-PMMA became visible in the surface potential image when humidity was above 90%. The surface potential difference between PS and PMMA blocks was around 0.4V. This result is consistent with the surface potential changes in LB films of PMMA with different tacticity4. A partial swelling of PMMA at high humidity leads to changes of the molecular dipole arrangement of this polymer and the related variations of surface potential. This effect was also observed in surface potential images of PS and PMMA blends with different composition. Furthermore, we performed KFM studies of two important industrial polymers: the blend of intrinsically conducting polymer poly (3,4-ethylenedioxythiophene) with polystyrene sulfonate acid (PEDOT:PSS) and perfluorinated polymer (Nafion), which is used in ion-exchange membranes. In both cases, the surface potential images, which were recorded in humid air, revealed hydrophilic PSS domains and water channels in Nafion. 1. S. Magonov, J. Alexander, Application Note “Exploring measurementsof local electric properties,” Agilent Technologies, Chandler, AZ (2008).2. J. Alexander, S. Magonov, M. Moeller JVST B27, 903 (2009).3. S. Magonov, J. Alexander, S.-H. Jeong, N. Kotov J. Nanosci Nanotechn, (2009) submitted.4. J.-J. Kim, S.-D. Jung, W.-Y. Hwang, ETRI Journal 18, 195 (1996)
10:00 AM - U6.2
Scanning Torsional Thermal Microscopy.
Michael McConney 1 , Kyle Anderson 2 3 , Hao Jiang 1 , Jesse Enlow 1 , Rachel Jakubiak 1 , Timothy Bunning 1 , Vladimir Tsukruk 2 3 Show Abstract
1 Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright Patterson Air Force Base, Dayton, Ohio, United States, 2 School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States, 3 School of Polymer, Textile and Fiber Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
Scanning Torsional Thermal Microscopy (STThM) utilizes thermal bimorph bending to create a torsion motion that can be measured independently of the normal height data caused by atomic forces between the AFM probe and the surface. The thermal torsional cantilevers are fabricated by coating both sides of conventional v-shaped tapping probes with polymer via plasma-enhanced chemical vapor deposition (PECVD). PECVD films have proven to be an excellent thermal bimorph material with absolute linear thermal expansion coefficients of 2-3x10-4 K-1. Following the coating of the v-shaped cantilevers, an arm of the top side coating and the opposite arm of the bottom side coating were removed with focused ion-beam. This bimorph geometry creates a probe which twists upon heating and cooling. STThM is operated in tapping mode, the damping of the horizontal “tapping” amplitude is used for the feedback signal and the torsion signal from the photo-detector is monitored for thermal variations. This torsion based approach relies on equipment already typically provided by AFM manufacturers and has been demonstrated to have impressive thermal sensitivity.
10:15 AM - U6.3
Carbon Nanotube Tips for High Resolution Electrochemical AFM.
Anne Beker 1 , Amol Patil 3 1 , Frank Wiertz 2 , Hendrik Heering 2 , Giacomo Coslovich 4 1 , Rifka Vlijm 5 1 , Tjerk Oosterkamp 1 Show Abstract
1 Leiden Institute of Physics (LION), Leiden University, Leiden Netherlands, 3 Chemistry, University of Oxford, Oxford United Kingdom, 2 Leiden Institute of Chemistry (LIC), Leiden University, Leiden Netherlands, 4 Physics, Università degli Studi di Trieste, Trieste Italy, 5 Molecular Biophysics, TU Delft, Delft Netherlands
Investigation of electrochemical phenomena using electrodes of nanometer size has been receiving increasing attention. Reducing the geometric dimensions increases the relative mass transport rate which yields high current densities coupled with small capacitances and reduced ohmic drop.We present a reproducible fabrication method of a high aspect ratio electrochemical Atomic Force Microscope (AFM) tip. Electrochemical nanoprobes were fabricated from a single polymer insulated multiwalled carbon nanotube (MWNT) mounted on a tapping mode AFM cantilever, using a home-built nanomanipulator1 . An electrochemically active length of carbon nanotube was exposed by laser ablation of the insulating polymer2. Characterization of these probes is done by cyclic voltammetry of Ferrocenemethanol in an aqueous solution. and by Finite Element Analysis. The fabricated nanoelectrodes were found to be stable and yielded an interfacial electron transfer rate constant (k0) of 1.073 ±0.36 cm/s for Ferrocenemethanol, at the surface of a clean single MWNT3.This versatile probe allows for a deeper understanding of electron transfer processes at the liquid-solid interface. Furthermore Carbon nanotube modified AFM cantilevers offer a solution to the problem of hindered diffusion of species to and from an electrochemically active substrate. Combining high topographical resolution and resistance to wear, these probes allow for studying a number of interesting systems: mapping proton or oxygen production or consumption from catalytic hotspots on a surface or monitoring enzyme products. Ongoing experiments aim at obtaining a first map of the activity of an oxygen reducing enzyme - part of the respiratory chain- on a conductive substrate.References E. C. Heeres, A. J. Katan, M. Hesselberth, A. F. Beker, D. J. van der Zalm, T. H. Oosterkamp, Rev. Sci. Instr., submitted. A. Patil, J. Sippel, G. W. Martin and A. G. Rinzler, Nano Lett., 2004, 4, 303-308 A. V. Patil, A. F. Beker, F. G. M. Wiertz, H. A. Heering, G. Coslovich, R. Vlijm and T. H. Oosterkamp, Nanoscale, submitted.
10:30 AM - U6.4
Tuning Fork Based Frequency Modulation AFM Bioimaging in Liquids With Multiple Probes.
Rimma Dekhter 1 , Galina Fish 1 , Sophia Kokotov 1 , Michael Kokotov 1 , Oleg Fedosyeyev 1 , Hesham Taha 1 , David Lewis 1 , Aaron Lewis 2 Show Abstract
1 , Nanonics Imaging Ltd., Jerusalem Israel, 2 Applied Physics, Hebrew University of Jerusalem, Jerusalem Israel
Atomic force microscopy with tuning fork feedback is the best method of AFM imaging known today. We now report the operation of normal force tuning fork feedback while the tuning fork is completely immersed in physiological media. This development allows the excellent abilities of high Q factor frequency modulation, that is the hallmark of tuning fork feedback, to be extended to biological AFM imaging with little or no damping and no need for Q control. The advance opens many new avenues including the first use of water immersion objectives with upright microscopes in BioAFM. It also permits near-field optical probes to be extended to live cell imaging overcoming the generally high force constants of such probes when they are cantilevered. In addition the non-optical feedback mechanism allows for transparently working with any optical technique at any wavelength including upright microscope Raman microprobes. Furthermore, the advance permits the implementation of multiprobe atomic force microscopy imaging in physiological media using the exposed probe tip of glass based AFM force sensors. This includes a variety of such probes with special import in biology. An example of the latter are nanopipette SPM probes for conductance and futuristic structurally correlated patch clamp applications.
10:45 AM - U6.5
Highly Customizable Scanning Probes Assembled with Nanomanipulation.
Peter Boggild 1 , Rajendra Kumar 1 , Volkmar Eichhorn 2 , Oezlem Sardan Sukas 1 , Florian Krohs 2 , Sergej Fatikow 2 Show Abstract
1 DTU Nanotech - Micro and Nanotechnology, Technical University of Denmark, Kgs. Lyngby Denmark, 2 Division of Microrobotics and Control Engineering, University of Oldenburg, Oldenburg Germany
We present here a proof-of-principle study of scanning probe tips defined by planar nanolithography and integrated with AFM probes using robotic nanomanipulation. The so-called ‘nanobits’ are 2–4 μm long and 120 nm thin flakes of silicon nitride and oxide, fabricated by electron beam lithography and standard silicon processing. Using an electrothermal microgripper mounted in a semiautomatic nanorobotic assembly system, the tiny flakes were detached from an array and fixed to a standard pyramidal AFM probe or alternatively inserted into a tipless cantilever equipped with a narrow slit. The nanobit-enhanced probes were used to image deep trenches, without visible deformation, wear or dislocation of the tips of the nanobit after several scans. In contrast to conventional scanning probe tip fabrication technologies, highly specialized shapes such as sidetips, double tips, soft tips, and high aspect ratio tips can be defined with no extra effort. This approach allows an unprecedented freedom in adapting the shape, size and even material of scanning probe tips to the surface topology, which opens a range of new possibilities in scanning probe microscopy and metrology of structures with special morphologies and mechanical properties, located in otherwise inaccessible locations, or with special requirements for functionalisation.
11:30 AM - U6.6
Multifunctional Ion Conductance Robot for Cross-disciplinary Studies at Nanoscale.
Andriy Shevchuk 1 Show Abstract
1 Medicine, Imperial College London, London United Kingdom
Progressive advances in scanning ion conductance microscopy (SICM) technique enabled us to convert ordinary scanning probe microscope (SPM) in to multifunctional nano-robot. As an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques. Also, it can visualize surfaces complexity of those makes them impossible to image by other SPMs. Ion conductance microscopy combined with a battery of powerful methods such as patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. The rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical will facilitate the study of integrated nano-behaviour in living cells in health and disease, material sciences, etc.References Hansma PK, Drake B, Marti O, Gould SA, Prater CB. The scanning ion-conductance microscope. Science 1989; 243: 641-643. Shevchuk AI, Frolenkov GI, Sanchez D, James PS, Freedman N, Lab MJ, Jones R, Klenerman D, Korchev YE. Imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy. Angew. Chem. Int. Ed Engl. 2006; 45: 2212-2216. Novak P, Li C, Shevchuk AI, Stepanyan R, Caldwell M, Hughes S, Smart TG, Gorelik J, Ostanin VP, Lab MJ, Moss GW, Frolenkov GI, Klenerman D, Korchev YE. Nanoscale live-cell imaging using hopping probe ion conductance microscopy. Nat. Methods 2009; 6: 279-281.
11:45 AM - U6.7
Ultrastable Atomic Force Microscope in Ambient Conditions.
Allison Churnside 1 3 , Gavin King 3 4 , Thomas Perkins 2 3 Show Abstract
1 Physics, University of Colorado, Boulder, Colorado, United States, 3 , JILA, Boulder, Colorado, United States, 4 Physics, University of Missouri, Columbia, Missouri, United States, 2 Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, United States
Instrumental drift remains a critical issue in scanning probe microscopy that limits tip-sample stability, registration and the signal-to-noise ratio during imaging. Here we show that by scattering a focused laser off the apex of commercial AFM tips, we can locally measure and thereby actively control a tip’s 3D position to < 0.4Å (Δf = 0.01–10 Hz) in ambient conditions. A second laser similarly stabilizes the sample substrate leading to a local, optically-based positional reference frame. These lasers operate in addition to the standard optical lever laser, which senses cantilever deflection. Using this optically-based reference frame, we demonstrate atomic-scale (≈1 Å) tip-sample stability and registration over 10s of minutes with a series of contact mode AFM images of 5-nm gold spheres on glass. These images were acquired at room temperature in air. We can now apply the ultrastable AFM to biological molecules in fluid. Our initial biological work uses force spectroscopy to study the folding and refolding of proteins (bacteriorhodopsin). We can independently measure the absolute height of the tip over the surface (using the new laser) and the force exerted on it by the protein (using the standard optical lever). We can also stabilize the tip-sample separation to observe dynamics over long times (>100 s). In summary, local optical stabilization of AFM extends atomic-scale control, previously restricted to cryogenic temperatures or ultra-high vacuum, to a wide range of operating environments, and we expect that it will find many applications in measurement and manipulation of single biological molecules.
12:00 PM - U6.8
AFM Atomic and Subnanometer Resolution in Non-UHV Conditions: A Critical Survey With Emphasis on Solids and Biological Systems.
Yang Gan 1 Show Abstract
1 School of Chemical Engineering & Technology, Harbin Institute of Technology, Harbin, Heilongjiang, China
Despite the enormous success and wide application of AFM in various areas, many researchers have still been conservative about the capability of AFM to achieve atomic and molecular resolution in ambient conditions. This scenario was formed, historically, due to numerous inappropriate declarations of obtaining so-called atomic and molecular resolution AFM topographs. In the last 15 years, reports of true atomic and molecular resolution in ambient conditions were by no means scarce; nevertheless, some have been overlooked and are rarely known, even to an AFM specialist! The AFM community could hardly benefit from this overlook because an AFM user, trained with this biased philosophy, might be frustrated and regard the challenge of achieving trustworthy high resolution as an impossible task. Instead, one needs to appreciate the power of AFM as an atomic and molecular scale analytical technique that is capable of producing reliable and reproducible topographs with various operation modes; at the same time, one needs to learn that AFM, as a local probe technique, is particularly vulnerable to artifacts. An experienced AFM user should be able to identify artifacts, avoid artifacts, and live skeptically with artifacts if necessary.This presentation will review critically the achievements of atomic and subnanometer resolution in non-UHV conditions by AFM. The concept of resolution is discussed after a brief introduction of various AFM operation modes. Various types of tip-surface forces, particularly the forces prominent in liquid and in air, are introduced. Different viewpoints on the conditions for achieving high resolution are discussed. The important issues of reproducibility and artifacts are discussed in depth, with some examples taken from the latest literature. Finally, the challenges of AFM as a trustworthy high resolution technique are discussed. (References: Y. Gan, Atomic- and Subnanometer Resolution in Ambient Conditions with Atomic Force Microscope, Surface Science Reports, 64 (2009) 99-121.)
12:15 PM - U6.9
Nanowires as AFM Cantilevers: A Detection Scheme to Gently Image and Interact with Soft Materials in Fluids.
Babak Sanii 1 , Paul Ashby 1 Show Abstract
1 Molecular Foundry, Lawrence Berkeley National Lab, Berkeley, California, United States
Performing AFM on soft materials in fluids (e.g., living cells) is challenging due to their ready deformation by the tip. The thermal force-noise of the cantilever is the principal limitation to reducing sample deformation and minimizing a cantilever's cross- section reduces its noise significantly. However, the minimum size of the cantilever is currently limited by a conventional deflection detection scheme, which requires a large surface area for laser specular reflection. Here we develop an optical technique to use nanowires as cantilevers, and show that we achieve a force noise in water that is orders of magnitude gentler than conventional AFM. This is a significant milestone towards non-invasive scanning probe imaging of biological processes on the surfaces of vesicles and cell membranes.
U7: Single-Molecule SPM
Thursday PM, April 08, 2010
Room 3000 (Moscone West)
2:30 PM - **U7.1
Nanoscale Chemical Spectroscopy With the Atomic Force Microscope.
Craig Prater 1 , Alexandre Dazzi 3 , Konstantin Vodopyanov 2 , Debra Cook 1 , Kevin Kjoller 1 Show Abstract
1 , Anasys Instruments, Santa Barbara, California, United States, 3 Laboratoire de Chimie Physique, Universite Paris-Sud, Orsay France, 2 E.L. Ginzton Laboratory , Stanford University, Stanford, California, United States
The ability to unambiguously identify arbitrary material under the tip of an AFM has been identified as one of the "Holy Grails" of probe microscopy. While the AFM has the ability to measure mechanical, electrical, magnetic and thermal properties of materials, the technique has lacked the robust ability to characterize and identify unknown materials. Infrared spectroscopy is a benchmark technique routinely used in a broad range of sciences to characterize and identify materials on the basis of specific vibrational resonances of chemical bonds. We have successfully integrated the capabilities of AFM with IR spectroscopy to allow chemical characterization on the micro and nanoscale. This technique enables the ability to obtain a high quality IR spectrum at an arbitrary point in an AFM image and/or automatically map the spectra at an array of points on a sample to enable chemical mapping. In this presentation, we will share the details of the measurement technique, along with application examples on polymer thin films, multilayers and blends, along with measurements on plant cells and bacteria.
3:00 PM - U7.2
Mapping Defects in Self-assembled Monolayers of Alkylsilanes Through Chemical Amplification.
Byron Gates 1 , Hanifa Jalali 1 , Yuanyuan Gong 1 Show Abstract
1 Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada
This talk will describe the ability to probe defects in self-assembled monolayers (SAMs) of alkylsilanes coating the surfaces of oxides. Insulating materials, such as thick layers of silicon dioxide, present a unique challenge for investigating the quality of these monolayers. A proper determination of the quality of the SAMs requires techniques to probe the defects in the surface modification. A chemical amplification technique can be used to highlight the defects in alkylsilane-based SAMs. The chemical amplification is necessary to increase the sensitivity of surface specific microscopy or spectroscopy techniques for assessing the quality of the SAMs. This amplification proceeds through a process of site selective material deposition and growth at defects in the surface modification. The deposited material further modifies the surface topography and local conductivity. These alterations to the surface are sufficient to improve the limits of detection for assessing the quality of SAMs. Highlights will include how this technique can be used to monitor the development of techniques for decreasing the density of defects in SAMs.
3:15 PM - U7.3
Electric Glue: Single-molecule Force Spectroscopy of Polymers With a Potentiostatically Controlled Surface.
Ann Fornof 1 2 , Matthias Erdmann 1 2 , Ralf David 1 2 , Hermann Gaub 1 2 Show Abstract
1 , Ludwig-Maximilians University, Munich Germany, 2 , Center for Nanoscience, Munich Germany
Single-molecule force spectroscopy has recently been employed to place molecules at desired locations as well as to measure the deposition events in single molecule cut and paste (SMCP).(1) The control over the pick-up and drop-off in the SMCP technique is dictated by passive hierarchical forces from the hydrogen bonding of DNA. Here we describe the active control of the adhesion (electrosorption) and interaction (physisorption) of DNA via the electric potential of a working electrode surface.(2) The DNA-electrode interaction is dictated by the externally controlled potential and modulated with differing surface and solution conditions. The influence of backbone charge on the DNA-electrode interaction was clarified by using two other biologically relevant polymers, PEG and a positively charged ionene. The measurement and control of the adhesion of single molecules to an electrode will be discussed.(1) Kufer, S.; Puchner, E.; Gumpp, H.; Liedl, T.; Gaub, H. Science, 2008, 319, 594.(2) Erdmann, M.; David, R.; Fornof, A.; Gaub, H. Nature Nanotechnology Accepted, 2009.
3:30 PM - U7.4
Molecular Cartography: Combined Topographical and Chemical Imaging Using AFM and Mass Spectrometry.
Olga Ovchinnikova 1 2 , Gary Van Berkel 1 2 Show Abstract
1 Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, 2 Department of Physics and Astronomy, University of Tennessee, Knoxville, Knoxville, Tennessee, United States
There exists a clear need to extend the limits of our understanding of chemical and physical phenomena of materials and biosystems at the nanoscale. Currently available techniques usually face a trade-off between spatial resolution and chemical information. Here we present the results of a novel technique that combines the spatial resolution of scanning probe microscopy with the analytical capabilities of mass spectrometry. This approach, which we refer to as atmospheric pressure hybrid proximal probe topography chemical imaging, uses a heated AFM probe to thermally desorb ma