Development of Temperature-Controlled High-Speed AFM

Wednesday, February 15, 2017 detector which converts the photon flux into a digital signal which is then displayed. To boost optical signatures many groups have shown that the close proximity of fluorescent species to fluorophores, significantly amplifies the fluorescence signatures many fold, as much as 103 in the near-field, a technology described as Metal-Enhanced Fluorescence by the Geddes labs in years past.1 However, hidden within these close-range near field fluorophore-metal interactions is an induced plasmonic current, directly proportional to the excitation irradiance and the concentration of the fluorophores present in the near-field, < 20 nm. The current can be read directly, opening up huge opportunities for both the amplification and the direct detection of fluorescence, i.e. digital fluorescence, such as in digital immunoassays DNA detection and in fluorescence microscopy. The direct measurement of fluorescence is likely to find profound applications and implications in the biosciences and promises to change both the way we think and use fluorescence spectroscopy today. In this presentation, we subsequently present our recent findings and demonstrate the role of different metallized substrates and the role of electron donors and quenchers on the magnitude of the induced Plasmonic Current. Reference ‘‘Metal-Enhanced Fluorescence,’’ Edited by Geddes, C.D., John Wiley and Sons, New Jersey, June 2010, 625 pgs, ISBN: 978-0-470-22838-8.

Force Spectroscopy and Scanning Probe Microscopy II 2887-Pos Board B494 Development of Temperature-Controlled High-Speed AFM Hirohide Takahashi1, Atsushi Miyagi1, Lorena Redondo-Morata2, Simon Scheuring1. 1 Weill Cornell Medicine, New York, NY, USA, 2Inserm, Marseille, France. High-speed atomic force microscopy (HS-AFM) has contributed important novel insights into the dynamics of biological samples such as lipid bilayers, membrane proteins, molecular motors and living cells at nanometer lateral and sub-second temporal resolution. Lipid bilayers are found in several different temperature-dependent states, termed phases; the main phases are solid and fluid phases. The transition temperature between solid and fluid phases is lipid composition specific. Under certain conditions some lipid bilayers adopt a so-called ripple phase, a structure where solid and fluid phase domains alternate with constant periodicity. Because of its narrow regime of existence and heterogeneity, ripple phase and its transition dynamics remain poorly understood. Here, a temperature control device is developed and integrated to high-speed atomic force microscopy (HS-AFM) allowing to observe dynamics of phase transitions from ripple phase to fluid phase reversibly in real time. Based on HS-AFM imaging, the phase transition processes from ripple phase to fluid phase and from ripple phase to metastable ripple phase to fluid phase could be reversibly, phenomenologically, and quantitatively studied. The results show phase transition hysteresis in fast cooling and heating processes, while both melting and condensation occur at 24.15
C in quasisteady state situation. A second metastable ripple phase with larger periodicity is formed at the ripple phase to fluid phase transition when the buffer contains Ca2þ. The presented temperature-controlled HS-AFM is a new unique experimental system to observe dynamics of temperature-sensitive processes at the nanoscopic level. 2888-Pos Board B495 High-Speed AFM Reveals Advanced Details on Dynamic Behavior of Antibody Norito Kotani, Ramanujam Kumaresan, Yoko Kawamoto-Ozaki, Takashi Morii, Takao Okada. Biomolecule Metrology, Research Institute of Biomolecule Metrology, Tsukuba, Japan. High-Speed Atomic Force Microscope (HS-AFM) can observe the dynamic behavior of biomolecules in solution, as a movie (1, 2). HS-AFM also can be used to image directly with high resolution in nano-scale order, without any chemical fixing of the samples or fluorescence. Antibody IgG and IgM are important proteins in Immune system. We have observed dynamic behavior of IgG and IgM in solution using HS-AFM. ‘‘Y’’ shape of IgG was imaged clearly, and Fab and Fc regions were well distinguished. IgM was imaged to contain five monomers, and each monomer has two antigen binding sites By HS-AFM imaging at video rate, we revealed that the Fab regions moved in torsional direction like swinging arms, and the soft structure of hinge regions

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facilitate this dynamic behavior. Using these soft arms, antibodies swivel in solution, groping for the antigens at surface, and the flexible nature of hinge region facilitates the binding to the antigen. We analyzed the Fab swivel movements as random walks, and estimated the flexibility of the IgG hinge region (3). Further, we imaged antibody-antigen interaction. The antibodies exhibited random flexible movements, as the two Fab regions bind to the antigen surface, leaving the Fc region to swing in all directions. Also, our studies exhibited that the binding occurs either in a standing posture, or in a low-lying posture. HS-AFM can directly observe dynamic behaviors of biomolecules as movie in solution, and reveal functions in regard to mechanical aspects. Direct and dynamic images have advantages to elucidate the properties of proteins and revealing the functional mechanisms. 1. T. Ando et al., Proc. Natl. Acad. Sci. U.S.A. 98, 12468-(2001) 2. N. Kodera et al., Nature 468: 72-(2010) 3. J. Preiner et al., Nature Communications 5: 4394-(2014). 2889-Pos Board B496 Non-Raster High-Speed AFM Imaging of Biopolymers Brett Hartman1, Sean Andersson1, William Nagel2, Kam Leang2. 1 Mechanical Engineering, Boston University, Boston, MA, USA, 2University of Utah, Salt Lake city, UT, USA. High-speed atomic force microscopy (HS-AFM) has advanced significantly in recent years with a few commercial instruments now able to image at rates between one to ten frames per second. These advances have allowed HSAFM to visualize biological molecules as they move. Despite these advances, however, there are several limitations in terms of utility as well as many systems of interest whose native speeds remain beyond current instruments, such as the motion of a cytoplasmic dynein with speeds on the order of 800 nm/s. Here, we describe a combination of non-raster scanning algorithms combined with a new high-speed three-axis scanning stage to achieve fast imaging on biopolymers and similar samples. The method combines a feedback-based algorithm that uses the data in real time to steer the tip along the biopolymer, decreasing imaging time by focusing the measurements on the interesting areas. The non-raster scanning algorithm is combined with the new high-speed, dual stage scanning platform. The scanner’s high resonances (in the tens of kHz range) is expected to achieve 30 frames per second or higher imaging rates. 2890-Pos Board B497 AFM Imaging of DNA G-Wires in Solution Krishnashish Bose, Anh Tuaˆn Phan. Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore, Singapore. Short G-rich DNA sequences have been reported to form G-wires, as observed by AFM imaging on Mica in air. However, till date such structures have not been observed directly in aqueous solution at the single-molecule level. Furthermore, the detailed structure of G-wires is not well understood. Understanding and controlling the formation of G-wires would have implications in their potential role in diseases and also nanotechnology applications. We developed a new sample preparation strategy and used ultra-short AFM cantilevers to overcome the problem of floating G-wires, which occurred with conventional protocols for AFM imaging of DNA in solution. Using our new protocol, we could image DNA G-wires in aqueous solution, and also resolved structural features down to 1 nm. We imaged G-wires formed by the Tetrahymena telomeric sequence d(G4T2G4) and other related sequences and found that these G-wires have a height of 2.950.3 nm, about 30-50% higher than what was reported previously. We also observed 4 nm periodic features which were either purely left-handed or zig-zag. For benchmark and comparison, we resolved the minor and major grooves of linear duplex DNA and obtained a height of 2.150.4 nm, which is very close to that of solution structure. Our work has opened up new possibilities for visualization of DNA G-quadruplexes in aqueous solution, which could allow investigation of their structure and interaction with other molecules. 2891-Pos Board B498 Extracellular Membrane Potential Measurement of Single Living Cells with Scanning Ion Conductance Microscopy Namuna Panday, Jin He. Physics, Florida International University, Miami, FL, USA. Recently, the existence of multiple micro-domains of extracellular potential around individual cells have been revealed by voltage reporter dye using