CeNTech and the University of Münster, Atomic Force F&E and Asylum Research are pleased to sponsor the Euro AFM Forum. The Forum is organized for researchers to share and exchange the cutting-edge research being done in Atomic Force Microscopy. It is open to anyone that currently uses AFM in their research or has an interest in AFM. The first day will feature presentations and posters from leading researchers on a variety of AFM topics from bio to semiconductors. The second day will be set aside specifically for a hands-on workshop and presentations.
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Registration
| Sept. 3: Early Registration & Reception 5:00pm,Sculpture Project Walking Tour, 6:00pm Sept. 4: Invited Talks and Posters Sept. 5: Equipment Workshop and Schedule Download the PDF of the Euro AFM Forum Program. Sept. 3: Early Registration & Reception 5:00pm, Sculpture Project Walking Tour, 6:00pm Investigation of Cellular and Molecular Processes by AFM Marcus Prass, Leif Riemenschneider, Stefan von Coelln, Steffi Prausse, Ken Jacobson & Manfred Radmacher, Institut für Biophysik, Universität Bremen Atomic force microscopy (AFM) can be used to investigate biological samples under physiological conditions and hence allows following the dynamics of biological processes. In addition, the application of forces allows elucidating the mechanical properties of the sample under investigation. At the cellular level the AFM allows measuring local mechanical properties of the cytoskeleton and thus gives new insights in processes like cellular locomotion or cell division. Changes in architecture of the cytoskeleton, or in activity of tension generating molecules (Myosin) can be picked up by mechanical measurements. Recently we were able to measure the local forces, which are generated at the leading edge of a migrating cell, so called protrusion forces. Despite the importance of cell migration in many processes, e.g. wound healing or metastasis, the actual process generating these forces is still unclear. Measuring directly the protrusion forces at the leading edge of a cell will help to understand the underlying biophysical processes. On the molecular scale, we could demonstrate that the AFM allows following conformational changes of enzyme molecules during their activity. By watching fluctuations in the molecules height under different buffer conditions, e.g. in the presence of substrate or inhibitors, we could clearly detect the dynamics of single molecules by AFM. Recently, we have employed single enzyme molecules, which are immobilized to the very apex of an AFM tip, to modify locally suitable samples. Thus we have demonstrated for the first time a new surface modification technique, which we called enzyme assisted nano-lithography.Titin Domains: A Prime Example in Single Molecule AFM Force Spectroscopy Measurements Dr. Wolfgang Linke, University of Münster The atomic force microscope (AFM) in its force-measuring mode has been extremely useful in elucidating the molecular basis of elasticity of various extracellular-matrix and cytoskeletal proteins. Titins constitute a super-family of giant elastic muscle proteins with a highly modular structure; their multiple tandem repeats of independently folded domains represent a common feature of proteins with structural and mechanical roles. The engineering of polyproteins consisting of repeats of identical titin immunoglobulin (Ig) domains has allowed detailed analysis of the mechanical properties of these domains. In the force-extension mode of the AFM the sequential unravelling of single Ig-like domains is observed as a sawtooth pattern of unfolding peaks, whereas in the more novel force-clamp mode Ig-domain unfolding appears as a stepwise increase in contour length. Force-quench experiments have proved particularly enlightening for our understanding of the refolding pathway of single modular proteins. Critique has been raised against a single-molecule approach to studying titin elasticity, in that the single molecule data may not directly be relevant to muscle elasticity, because structural constraints and/or homotypic binding or interactions with other proteins could modulate titin elasticity in vivo. A current goal in AFM-based titin mechanics therefore is the investigation of possible modifiers of titin elasticity, e.g., heat shock proteins, protein kinases, or intra/intermolecular bond formation. Studying protein mechanics under conditions better resembling those found in vivo will be a challenge for future AFM force spectroscopy work. Measuring Physical Properties and Interaction Forces at Microbial Surfaces by Force Spectroscopy using the AFM Fabien Gaboriaud, Laboratory of Physical Chemistry and Microbiology for the Environment In the last decade, remarkable advances have been made in applying force spectroscopy with the AFM instrument to quantify the interaction forces and physical properties of microbial surfaces [1]. Such physico-chemical characteristics play an important role in controlling interfacial phenomena such as microbial adhesion, biocorrosion, microbial infection or biofilm formation. In this presentation, I will present different applications of force spectroscopy for exploring microbial surfaces at the cell and molecular scales. In particular, we used force volume mode AFM to accurately locate the apical surface and periphery of the cell since, in contact mode, a component of the applied load laterally deforms the cell during the raster scan [2]. Furthermore, we combined force spectroscopy and advanced electrokinetic analysis to provide a detailed physico-chemical capture of the microbial interface in terms of coupled electrostatic and hydrodynamic characteristics, structural heterogeneities, and mechanical properties [3-6]. [1] Gaboriaud F. and Dufrêne Y. F. (2007) "Atomic force microscopy of microbial cells: Application to nanomechanical properties, surface forces and molecular recognition forces" Colloids and Surfaces B: Biointerfaces 54, 10-19. Single Molecule Protein-RNA Interactions Alexander Fuhrmann (1), Jan Schöning (2), Sebastian Getfert (3), Dario Anselmetti (1), Peter Raimann (3), Dorothee Staiger (2) and Robert Ros (1). 1. Experimental Biophysics and Applied Nanoscience / 2. Molecular Cell Physiology / 3. Condensed Matter Theory, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany Post-transcriptional regulation represents an important mechanism to control gene expression in higher plants. It implies processes at various hierarchic levels including pre-mRNA maturation, mRNA transport, translation and breakdown. The major players are RNA-binding proteins that, by binding to defined RNA sequences, influence and control the fate of an mRNA molecule either directly or indirectly through protein-protein interaction. The abundance of the Arabidopsis thaliana glycine rich RNA binding proteins ATGRP7 and ATGRP8 is under control of the endogenous clock. ATGRP7 is part of a negative feedback loop through which the protein regulates the circadian oscillations of its own mRNA at the post-transcriptional level. ATGRP7 and ATGRP8 combine an N-terminal RNA recognition motif with a C-terminal region enriched in glycine repeats with some interspersed serine, tyrosine and arginine residues [1]. We are using atomic force microscopy (AFM) based force spectroscopy to investigate the interaction of ATGRP7 and ATGRP8 with RNA target sequences at the single molecule level [2]. By introducing point mutations in the proteins as well as in the RNA sequences we are able to discriminate sequence-specific from nonspecific electrostatic interactions. We apply and improve the concept of the heterogeneity of chemical bonds [3] for analyzing our single molecule force spectroscopy data, resulting in quantitative characterization of the protein-RNA interactions in terms of rate constants and dissociation lengths. Furthermore, our analysis technique enables us to get insights into the molecular binding mechanism. Session 2 Abstracts Expanding the Temporal and Spatial Scales in Scanning Force Microscopy László Grama, Attila Nagy, Árpád Karsai, Pasquale Bianco, András Kengyel, Tamás Huber, Zsolt Mártonfalvi, Balázs Kiss, Ünige Murvai, Margit Benke, Brennan Decker and Miklós S.Z. Kellermayer. Dept. Biophysics, University of Pécs, Faculty of Medicine, Szigeti út 12., Pécs, H-7624 Hungary The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years. However, it lacks the time resolution and functional specificities offered by other methods. An important aim of our research is to expand the spatial and temporal scales of AFM either by combining it with fluorescence or by employing alternative scanning schemes on unique biomolecular systems. To simultaneously exploit the advantages of AFM and fluorescence, we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument is a stage-scanning device in which the mechanical and optical axes are co-aligned to achieve spatial synchrony. At each point of scan the sample topography (AFM) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. Using the technique correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution. To enhance the temporal resolution of AFM for the purpose of following fast biomolecular events, we applied a simply modified technique called scanning force kymography. Using the method we monitored the growth, on mica surface, of individual amyloid fibrils with near-subunit (~1 nm) spatial and subsecond (~300 ms) temporal resolution. Amyloid fibril assembly was polarized and discontinuous. Bursts of rapid, up to 300 nms -1 growth phases that extended the fibril by ~8 nm or its integer multiples were interrupted with pauses. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The employed scanning force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers. Myofibrillar and Cellular Structure of Cardiac Myocytes: A Combined Confocal and Atomic Force Microscopy Study Nicholas A. Geisse, Sean P. Sheehy, Kevin Kit Parker, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138 In this study, we have used microcontact printing techniques to control the shape of neonatal rat ventricular myocytes (NNRVMs) in culture. By controlling the boundary conditions of these cells, cardiac myocytes of varying length-to-width ratios were fabricated, similar in shape to those observed in the failing heart. Utilizing three-dimensional confocal microscopy and Atomic Force Microscopy (AFM), we have demonstrated that NNRVMs cultured on fibronectin (FN) islands with a surface area of ~2500 µm2 form a pseudo-two-dimensional (2D) shape. The alignment of sarcomeres in these cells is highly reliant on extracellular boundary conditions, and changes from a longitudinal orientation to a bi-axial one as cardiac myocytes become shorter and wider. We also show that NNRVMs of varying shape, with consistent areas, have similar volumes when measured by AFM, suggesting a growth control mechanism related to the cell-ECM contact area. Finally, we demonstrate that mechanical indentation with an AFM tip can cause a release of Calcium inside the cell as measured by fluorescent Calcium indicators and epifluorescent microscopy. The magnitude of the force required for this release is sensitive to the location that it is placed on the cell relative to the distribution of different cytoskeletal elements throughout the cell volume, suggesting a molecular mechanism for mechanotransduction in the cardiac myocyte. Single Molecule Studies on the Mechanical Activation of the Molecular Force Sensor Titin Kinase E. Puchner,*, F. Gräter (+), A. Alexandrovich (#), B. Brandmeier (#) and Molecular force sensors are the core of signaling pathways monitoring mechanical units or reporting mechanical load. It has been shown, that titin, a giant protein located in muscle sarcomer, contains a kinase domain that controls muscle gene expression and protein We investigated the mechanical properties of titin kinase be means of single molecule force spectroscopy. Our results show, that this enzyme complies with the requirements of a force sensor. Re-enacting the natural setting on a single molecule level evidences, that during a mechanical activation cycle ATP is able to bind to the titin kinase. Our results allow new insight into the function and dynamics of this intriguing enzyme and characterize the first, and until now missing part of its mechano-chemical signaling pathway. Dual Frequency Atomic Force Microscopy R. Proksch, D. Bocek, C. Callahan, J. Cleveland, T. Day, J. Li, and A. Moshar, Asylum Research.In a new imaging mode (“Dual AC”), the cantilever is driven at or near two of its resonant vibrational modes. The amplitude and phase signals measured at the different frequencies show very different contrast on a variety of samples. [1] In one example shown here, the driven second resonance mode phase shows strikingly different contrast from the same fundamental resonance signals on a Type I collagen sample imaged in air. A side-by-side comparison shows the fundamental resonance phase data painted as an overlay on the AFM topography (left) and second mode phase data overlaid on the AFM topography (right). The red arrows indicate suspended fibers that absorb second resonance energy but show no fundamental resonance contrast. There are patches of contamination (green arrows) on the glass substrate (blue arrows). Again, the second mode phase shows a distinct difference between the patches and the glass not visible in the first. In addition to imaging results, we will discuss some insights into the origins of the contrast we have made through numerical simulations and by performing amplitude and phase vs. tip-sample distance curves we have performed above different regions of various samples in air and in liquid. [1] R. Proksch. Multifrequency, repulsive-mode amplitude-modulated atomic force microscopy. Applied Physics Letters 89, 113121 (2006).
Image caption left: Fundamental resonance phase overlaid on the topography, 4µm scan. Image caption right: Second resonance phase overlaid on the topography. Session 3 Abstracts Charge Writing in Thin Film Polymer Electrets Dr. Nikolaus Knorr, Silvia Rosselli, Tzenka Miteva, Akio Yasuda, Gabriele Nelles One of the great challenges of nanotechnology is the use of an AFM like probe system for high data density storage. IBM is working on a device called “millipede” that writes bits in parallel by thermomechanical probe indentation in polymer thin films. We have investigated the viability of different thin film polymer electrets as probe charge storage materials. Charge spot bits are written by voltage pulse application to the conducting probe which is in contact to the polymer film and read out by electrostatic force microscopy (EFM) imaging. We report on the impact of writing parameters (pulse height and width), material type, film thickness, probe geometry, and writing conditions (ambient, dry air) on key storage parameters like charge spot strength and size, retention time, and rewritability. Piezoelectric Dilute Magnetic Semiconductors: Towards Functional Multi-ferroics L. Belova, M. Kapilashrami and K.V. Rao Dept. of Materials Science and Engineering, Royal Institute of Technology, Stockholm, Sweden. Zinc oxide is a multifunctional dielectric material [1,2] which has caught a lot of attention in recent years among the researchers in the fields of semiconductor- and optoelectronic materials, both as bulk material and as transparent thin films. ZnO is an II-VI material with a band gap near the UV range (eg ~3,3 eV) at room temperature showing properties similar to GaN (eg ~3,4 eV) [3]. Being a large band-gap material (direct band-gap), ZnO has a large breakdown voltage meaning that the material can work at large electric fields and at higher temperatures. As the natural structure of ZnO is the hexagonal Wurtzite structure, it has a low centre symmetry, which results in the existence of spontaneous polarisation along the c-axis, so to say it displays piezoelectric properties. This means that it can produce spontaneous polarisation when the material is mechanically stressed and vice versa experience a mechanical strain per unit of applied field. It has earlier been shown that doping ZnO with transition metals (TM) i.e. Cu, Mn, V results in above room-temperature ferromagnetism [4,5], where the TM acts as the spin carrier. As well has it also been shown that moderate doping of ZnO enhances the piezoelectric property of ZnO [6].Making ZnO ferromagnetic by doping it with transition metals yet sustaining its transparency and combining this property with its piezoelectric properties opens up a whole new field of application for this class of material, also known as Multiferroics materials. In this current study we investigate the tunability of the magnetic properties of ZnO, from being a diamagnetic material to an above room-temperature ferromagnetic material by doping it with Mn (up to 5 at.%) and varying deposition parameters. Further we study piezoelectric properties of the tailored doped ZnO with the use of AFM. We will discuss enhancement of piezoelectric properties of Zn1-xMnxO with doping and interplay of piezoelectric and magnetic properties of these materials making them perfect candidates for transparent multiferroic device components. References Chemical Probing of Soft Materials by AFM: Challenges, Hopes and Limitations Dr.Holger Schönherr, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands, h.schonherr@tnw.utwente.nl Tailored micro- and nanopatterned organic and polymer surfaces play an essential role among others in biosensors and for the fabrication of sophisticated biointerfaces, e.g., for the study of cell - surface interactions. Here the relevant distances and length scales of ligand clustering in multivalent recognition or those involved in protein clustering in focal adhesion of certain types of cells on the one hand, and the size of biological entities, such as bacteria or cells, on the other hand, span an enormous range. Clearly, new approaches towards (bio)chemical patterning as well as in particular laterally resolved surface compositional and structural analysis are central challenges that must be addressed in this context. In this contribution our efforts in the development of approaches to carry out highly localized grafting reactions and to characterize the chemical composition of the nanopatterns obtained will be summarized. Using atomic force microscopy (AFM) with chemically modified tips, surface chemical composition can be probed, as shown, in the critical sub-100 nm regime [1]. However, despite the success of this methodology in some areas to be discussed, including probing of surface treated polymers or the measurement of single molecule interactions, properties, and dynamic processes, important practical issues remain unsolved or until recently difficult to overcome. These issues include for instance the reliable calibration of AFM lateral force microscopy, as widely used for the analysis of patterned layers or nanostructured obtained by nanolithography [2,3]. The challenges, hopes and limitations of "chemical probing of soft matter by AFM" will be critically discussed in the light of recently developed approaches to realize nanostructured biointerfaces [4]. References: [1] G. J. Vancso, H. Hillborg, H. Schönherr Adv. Polym. Sci. 2005, 182, 55. Local Anodic Oxidation on GaAs with an Atomic Force Microscope D. Graf, T. Ihn, and K. Ensslin We demonstrate the fabrication of high-quality electronic nanostructures by scanning probe-based lithography. The application of a negative voltage to a conductive tip of an atomic force microscope in a humid environment leads to the local oxidation of the surface underneath [1]: the tip serves as cathode, the sample as anode, and the electrolyte is the water film formed naturally on top of the sample surface. This nano-lithography technique is competitive with electron beam lithography for fabricating electronic devices on a nanometer scale. Using modulated instead of continuous voltages will avoid traps of ionic charges accumulating in the oxide and also reduce the charge diffusion in the water meniscus forming between the tip and sample [4]. As a result we find an improved lateral resolution of the patterned oxide i.e. a better aspect ratio of the lines [5]: the self-limiting growth is overcome due to the AC electric field which neutralizes the reaction products. [1] H. C. Day and D. R. Allee, Appl. Phys. Lett. 62, 2691 (1993) Session 4 Abstracts Scanning Nanopipettes: A New Tool for NanoBio-Analytics Tilman E. Schäffer (1), Matthias Böcker (1), Anke Engbert (1), Johannes Rheinlaender (1), Steffen Muschter (1), Eva Sicking (1), Joachim Wegener (2), (1) Institute of Physics and Center for Nanotechnology, University of Münster, Germany (2) Institute for Biochemistry, University of Münster, Germany Nanopipettes with opening diameters of 100 nm or less provide probes for the locally resolved measurement of ionic currents on the nanometer scale. The nanopipettes are filled with an electrolyte and are scanned over a sample surface using a scanning ion conductance microscope (SICM). We used SICM to study the ion permeability of tissue-like cell layers with lateral resolution. For MDCK-II cells, we measured a larger ion conductance along the cell periphery in areas of cell-cell contacts, compared to that along the cell bodies. This suggests that ions mainly pass through the paracellular cleft between adjacent cells but not through the cellular plasma membrane. In a second application, we created a nanopore of adjustable size by pushing the tip of a nanopipette into a deformable polymer sample. Two different regimes were identified: a “current-squeezing” regime at large pipette-sample distances and a “seal-forming” regime at close pipette-sample distances. By controlling the pipette-sample distance, the effective opening diameter of the pipette tip can be adjusted. Possible applications of such a nanopore of adjustable size are sensing and sizing of single molecules. How Can Atomic Force Microscopy Help to Fight Against Bacterial Infections? Böhling A., Gutsmann S., Brauser A., Gutsmann T., Research Center Borstel There is an ongoing battle between the immune system and invading pathogens, e.g. microorganisms such as bacteria, or phathogenic factors, i.e. structures released from the microorganisms. We focused on two important aspects of the Gram-negative sepsis, which causes about 50% of all septic diseases. Gram-negative bacteria are surrounded by a cell envelope, consisting of the cytoplasmic or inner membrane, a thin peptidoglycan layer, and an additional barrier, the outer membrane (OM). The lipid composition of the OM is asymmetric: the inner leaflet is composed of a mixture of phospholipids and the outer leaflet of glycolipids, in most cases lipopolysacchrides (LPS). The OM and in particular the LPS plays a dual role: (i) All antimicrobial agents attacking Gram-negative bacteria have to permeate through the OM or to destroy it; (ii) When LPS is released from the OM during cell division or induced by antibacterial agents, it interacts with immune cells and simulates them to release mediators, e.g. proinflammatory cytokines. The activation of the immune cells can finally lead to sepsis. To get insight into the molecular interaction mechanisms between lipid membranes and peptides or proteins, the complex biological system of human cells or bacteria has often to be reduced. Therefore, membranes are reconstituted from purified lipid preparations to perform biophysical experiments on simplified models. We used the atomic force microscope to investigate the structure and function of these lipid membranes and their interaction with antimicrobial peptides as well as with proteins being involved in the LPS-induced signal transduction in human immune cells. Spring Constant Determination with Commercial AFMs in Buffers of Various Viscosity Tobias Pirzer, Institute of Medical Engineering and Physics Department, Technische Universität München The biggest uncertainty in the absolute determination of forces comes from measuring the optical lever sensitivity and the spring constant of the cantilever. The former uncertainty can be reduced by averaging over some indentation traces. The best determination of the latter is the topic of this talk. In commercial AFMs the spring constant is usually determined from the thermal power spectrum density by a (damped) simple harmonic oscillator (SHO) fit. We first compare the spring constants of AFM cantilevers in three commercial AFMs in low viscosity media like water and air. Then we turn to high viscous media (e.g. 5 M Phosphate buffer). There the deviations from the true spring constant can be more then 100% when utilizing the SHO fit. This comes because there is no analytical function for a SHO fit at low Q. We discuss the limits of the SHO fit and present the thermal noise method as an alternative to determine the spring constant with commercial instruments in buffers of various viscosity. Poster boards will be put up in the CeNtech on the first day of the conference. Presenters may place their poster on the boards that correspond to their poster number below anytime during the conference- on the evening of Sept. 3 or during the conference on Sept. 4. The official poster session will be from 5:30-7:00pm on Sept. 4. 1. Small Amplitude Oscillation Lateral Force Microscopy 2. Conductance Imaging of Cell-Cell Contacts with Scanning Ion Conductance Microscopy (SICM) 3. First Force Spectroscopy Measurements of Physiologic VLA-4 Integrin Activation by the Chemokine SDF-1 at the Single-molecule Level on a Living Cell 4. Topology of Casein Micelle in Their Natural Environment by Atomic Force Microscopy 5. KP-AFM Detection of Changes in the Spontaneous Polarization of P(VDF)-TrFE Thin Films by Thermal Stimulation 6. Resolution in Scanning Ion Conductance Microscopy: A Finite Element Analysis 7. CELL Softening Upon Thirst Hormone 8. AFM Investigations of Track Membranes—Nanostructures Formed by Replication of Track Membranes 9. Lipitades Peptides and Proteins in Model Membranes 10. Interactions of Biofunctionalized Nanoparticles with Artificial Membranes 11. Towards Nanomanipulation with Atomic Force Microscopy and Fluorescence Control 12. Semiconducor Nanostructures Investigated by Atomic Force Microscopy 13. Investigation on Semiconductor Quantum Dots by Using Atomic Force Microscopy 14. Oscillation Control of a Quartz Sensor in Scanning Probe Microscopy 15. Advanced High-speed AFM Stage for Precise Positioning 16. Refined Procedure of Evaluating Experimental Single Molecule Force spectroscopy Data 17. Simultaneous Measurement of Sample Topography and Electrostatic Potential by 18. Specific and Controlled Positioning of Single Biomolecules with Atomic Force Microscopy 19. PUNIAS: Protein Unfolding and Nano-Indentation Analysis Software 20. Ultrastructure of Trypanosoma Cruzi Revisited by AFM Sept. 5: Equipment Workshop and Lectures The equipment workshops will begin Wednesday, Sept. 5 at 9:00 at the CeNTech and at the University of Munster. A shuttle will be provided to and from the sessions being held at the Linke Lab at the University of Munster. Each workshop registrant will receive confirmation of their selected workshop topics via email and an updated schedule upon arrival at the conference. Lectures are open to anyone that wishes to attend. Lunch will be provided. Download the PDF of the current Workshop Schedule. | |
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