Bulletin of the American Physical Society
2008 APS March Meeting
Volume 53, Number 2
Monday–Friday, March 10–14, 2008; New Orleans, Louisiana
Session P14: High-Bandwidth Dynamic Atomic Force Microscopy |
Hide Abstracts |
Sponsoring Units: DBP BPS FIAP Chair: Brian Salzberg, University of Pennsylvania School of Medicine Room: Morial Convention Center 205 |
Wednesday, March 12, 2008 8:00AM - 8:36AM |
P14.00001: High-Bandwidth Atomic Force Microscopy Reveals A Mechanical spike Accompanying the Action Potential in mammalian Nerve Terminals Invited Speaker: Information transfer from neuron to neuron within nervous systems occurs when the action potential arrives at a nerve terminal and initiates the release of a chemical messenger (neurotransmitter). In the mammalian neurohypophysis (posterior pituitary), large and rapid changes in light scattering accompany secretion of transmitter-like neuropeptides. In the mouse, these intrinsic optical signals are intimately related to the arrival of the action potential (E-wave) and the release of arginine vasopressin and oxytocin (S-wave). We have used a high bandwidth (20 kHz) atomic force microscope (AFM) to demonstrate that these light scattering signals are associated with changes in nerve terminal volume, detected as nanometer-scale movements of a cantilever positioned on top of the neurohypophysis. The most rapid mechanical response, the ``spike'', has duration comparable to that of the action potential ($\sim $2 ms) and probably reflects an increase in terminal volume due to H$_{2}$O movement associated with Na$^{+}$-influx. Elementary calculations suggest that two H$_{2}$O molecules accompanying each Na$^{+}$-ion could account for the $\sim $0.5-1.0 {\AA} increase in the diameter of each terminal during the action potential. Distinguishable from the mechanical ``spike'', a slower mechanical event, the ``dip'', represents a decrease in nerve terminal volume, depends upon Ca$^{2+}$-entry, as well as on intra-terminal Ca$^{2+}$-transients, and appears to monitor events associated with secretion. A simple hypothesis is that this ``dip'' reflects the extrusion of the dense core granule that comprises the secretory products. These dynamic high bandwidth AFM recordings are the first to monitor mechanical events in nervous systems and may provide novel insights into the mechanism(s) by which excitation is coupled to secretion at nerve terminals. [Preview Abstract] |
Wednesday, March 12, 2008 8:36AM - 9:12AM |
P14.00002: Studying Chemical Reactions, One Bond at a Time, with Single Molecule AFM Techniques Invited Speaker: The mechanisms by which mechanical forces regulate the kinetics of a chemical reaction are unknown. In my lecture I will demonstrate how we use single molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bond via the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is of common occurrence in mechanically stressed proteins. While reduction is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction, the role of a mechanical force in modulating this chemical reaction is unknown. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by dithiothreitol (DTT). We find that while the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300 pN range. This result predicts that the disulfide bond lengthens by 0.34 {\AA} at the transition state of the thiol/disulfide exchange reaction. In addition to DTT, we also study the reduction of the engineered disulfide bond by the E. coli enzyme thioredoxin (Trx). Thioredoxins are enzymes that catalyze disulfide bond reduction in all organisms. As before, we apply a mechanical force in the range of 25-450 pN to the engineered disulfide bond substrate and monitor the reduction of these bonds by individual enzymes. In sharp contrast with the data obtained with DTT, we now observe two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulfide bond, causing a shortening of the substrate polypeptide by 0.76$\pm$0.07 {\AA}, and the second elongating the substrate disulfide bond by 0.21$\pm$0.01 {\AA}. These results support the view that the Trx active site regulates the geometry of the participating sulfur atoms, with sub-{\AA}ngstr\"om precision, in order to achieve efficient catalysis. Single molecule atomic force microscopy (AFM) techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site which cannot be resolved with any other current structural biological technique. Furthermore, our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property which has never been explored by traditional biochemistry. \newline \newline 1.-Wiita, A.P., Ainavarapu, S.R.K., Huang, H.H. and Julio M. Fernandez (2006) Force-dependent chemical kinetics of disulfide bond reduction observed with single molecule techniques. \textbf{Proc Natl Acad Sci} U S A. 103(19):7222-7 \newline 2.-Wiita, A.P., Perez-Jimenez, R., Walther, K.A., Gräter, F. Berne, B.J., Holmgren, A., Sanchez-Ruiz, J.M., and Fernandez, J.M. (2007) Probing the chemistry of thioredoxin catalysis with force. \textbf{Nature}, 450:124-7. [Preview Abstract] |
Wednesday, March 12, 2008 9:12AM - 9:48AM |
P14.00003: Imaging and Beyond with High Speed AFM. Invited Speaker: It is now possible to operate Atomic Force Microscopes (AFMs) at speeds of up to 6000 lines per second over scan ranges exceeding 10 microns. For a 100 x 100 pixel image this gives frame rates of 60 frames/second: faster than video rate. This has required small cantilevers, new scanners, new high voltage amplifiers, and a new scan control system. The small cantilevers are from SCL Sensor-Tech (Deutsch-Wagram, Austria). The new scanner is based on a sophisticated system of flexures that constrain the motion of each separate piezo stack to one dimension in a three-dimensional scanner. It has a scan range of 15 microns and a lowest resonance frequency of about 27 kHz. The new high voltage amplifier, built in collaboration with TechProject (Vienna, Austria), can deliver up to 8 amps over the entire output range from 0 to 150 volts with the challenge of having the piezo as a capacitive load. The new scan control system is built around a commercially available DAQ board in a Windows environment. One of the major challenges is now to move beyond imaging to Force-Volume imaging, which involves taking an array of force curves over a sample and then reconstructing a zero force image as well as a map of local mechanical properties. [Preview Abstract] |
Wednesday, March 12, 2008 9:48AM - 10:24AM |
P14.00004: AFM probes with integrated electrostatic actuators for fast, quantitative imaging and force spectroscopy Invited Speaker: In this talk, we summarize our efforts in developing novel AFM probes (FIRAT) with integrated sensing and actuation. These probes exploit recent advances in microscale sensor technology and open up the design space for AFM applications including fast imaging, quantitative material characterization and single molecular mechanics measurements. For fast imaging applications in air, probes with aluminum force sensing structures are surface micromachined on quartz substrates. Using 0.7-0.8$\mu $m thick, 40$\mu $m$\times $60$\mu $m clamped-clamped beams over 2.8$\mu $m of air gap, probes with resonance frequencies in the order of 1MHz and Q in the 5-15 range are obtained. These probes are actuated directly by electrostatic forces applied to the mechanical structure by rigid electrodes on the substrate shaped as optical diffraction gratings, enabling imaging bandwidths in the order of 100kHz. The integrated grating interferometer provides 10fm/$\surd $Hz level displacement sensitivity down to 3Hz. The surface micromachining approach used for probe fabrication lets one to precisely control the probe dynamics and overcome the difficulties associated with regular AFM cantilevers for applications such as time resolved interaction force (TRIF) measurements. Using FIRAT probes with over damped dynamics, clean TRIF signals are obtained while imaging the surface at regular speeds. This enables us to use a simple model to invert quantitative mechanical properties of a variety of polymers. For measurements on single molecules, membrane type FIRAT probes suitable for in liquid operation have been developed. These probes are made of dielectric materials with embedded actuation electrodes. Used only as actuators or both actuators and force sensors, these devices are shown to enable parallel force spectroscopy measurements. We also show that the spring constant of these probes can be electrically reduced to achieve higher force sensitivity while not affecting its noise performance and discuss the effect of hydrodynamic forces in these membrane type probes as compared to cantilever type probes for fast force spectroscopy measurements. [Preview Abstract] |
Wednesday, March 12, 2008 10:24AM - 11:00AM |
P14.00005: High-speed AFM for Studying Dynamic Biomolecular Processes Invited Speaker: Biological molecules show their vital activities only in aqueous solutions. It had been one of dreams in biological sciences to directly observe biological macromolecules (protein, DNA) at work under a physiological condition because such observation is straightforward to understanding their dynamic behaviors and functional mechanisms. Optical microscopy has no sufficient spatial resolution and electron microscopy is not applicable to in-liquid samples. Atomic force microscopy (AFM) can visualize molecules in liquids at high resolution but its imaging rate was too low to capture dynamic biological processes. This slow imaging rate is because AFM employs mechanical probes (cantilevers) and mechanical scanners to detect the sample height at each pixel. It is quite difficult to quickly move a mechanical device of macroscopic size with sub-nanometer accuracy without producing unwanted vibrations. It is also difficult to maintain the delicate contact between a probe tip and fragile samples. Two key techniques are required to realize high-speed AFM for biological research; fast feedback control to maintain a weak tip-sample interaction force and a technique to suppress mechanical vibrations of the scanner. Various efforts have been carried out in the past decade to materialize high-speed AFM. The current high-speed AFM can capture images on video at 30-60 frames/s for a scan range of 250nm and 100 scan lines, without significantly disturbing week biomolecular interaction. Our recent studies demonstrated that this new microscope can reveal biomolecular processes such as myosin V walking along actin tracks and association/dissociation dynamics of chaperonin GroEL-GroES that occurs in a negatively cooperative manner. The capacity of nanometer-scale visualization of dynamic processes in liquids will innovate on biological research. In addition, it will open a new way to study dynamic chemical/physical processes of various phenomena that occur at the liquid-solid interfaces. [Preview Abstract] |
Follow Us |
Engage
Become an APS Member |
My APS
Renew Membership |
Information for |
About APSThe American Physical Society (APS) is a non-profit membership organization working to advance the knowledge of physics. |
© 2024 American Physical Society
| All rights reserved | Terms of Use
| Contact Us
Headquarters
1 Physics Ellipse, College Park, MD 20740-3844
(301) 209-3200
Editorial Office
100 Motor Pkwy, Suite 110, Hauppauge, NY 11788
(631) 591-4000
Office of Public Affairs
529 14th St NW, Suite 1050, Washington, D.C. 20045-2001
(202) 662-8700