Mar 2, 2016 - Hao Yu1, Matthew G.W. Siewny1, Devin T. Edwards1, Aric W. Sanders2,. Thomas T. Perkins1,3. 1JILA, National Institute of Standards and ...
Wednesday, March 2, 2016 with nanometer resolution. However, it traditionally suffers from extremely low throughput. When parallelizing bead-tracking experiments, the need arises for fast algorithms without compromising accuracy and robustness. Here, we introduce a novel tracking method based on a 3D cross correlation of the bead image with a set of computer-generated reference images. We show that the phase shift of the cross correlation peak is proportional to the bead height. For testing the accuracy, we used Lorentz-Mie scattering theory to generate test data. We found that non-circular effects such as interlacing, small image artifacts and non-uniform illumination do not affect the tracking result. Our algorithm tracks over 100 beads in a 100x100 pixel region-of-interest on a multicore PC in real time. Its speed, accuracy and robustness may improve other techniques that rely on bead tracking such as optical tweezers and acoustic force spectroscopy. 2547-Plat Simultaneous Advanced Microscopies for Live Cell Signaling Dynamics Investigations Adelaide Miranda, Marco Martins, Pieter A.A. De Beule. Applied Nano-Optics Laboratory, International Iberian Nanotechnology Laboratory, Braga, Portugal. We present a new type of combined microscopy for the life sciences based on the integration of differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping Atomic Force Microscopy (AFM).[1] Our hardware platform creates the opportunity for simultaneous acquisition of membrane receptor recognition maps and fluorescence optical sectioning images of live cells, providing new experimental capabilities in cell signaling investigations. Simultaneous spatio-temporal generation of AFM and fluorescence microscopy data is made possible through time-independent illumination at low light intensities of the AFM cantilever that strongly reduces the interaction of AFM cantilever motion with fluorescence excitation light as compared to existing hardware integration platforms based on confocal laser scanning microscopy and AFM. Namely, fluorescence excitation light has the capability to induced cantilever heating and subsequent sample heating through heat conduction. We identify several system specific noise-sources that can interfere with live cell investigations and describe solutions to mitigate them. [1] Miranda, A., Martins, M. & De Beule, P. A. A. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy. Rev. Sci. Instrum. 86, 093705 (2015). 2548-Plat Video-Based Force Detection in Optical Tweezers to Measure DNA Translocation through SI-NX and Lipid-Coated Nanopores Andy Sischka1, Sebastian Knust2, Lukas Galla2, Andreas J. Meyer2, Andre Spiering2, Michael Mayer3, Adam R. Hall4, Peter Reimann2, Karsten Gall1, Dario Anselmetti2. 1 Ionovation GmbH, Osnabrueck, Germany, 2Bielefeld University, Bielefeld, Germany, 3Department of Chemical Engineering and Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 4 Department of Biomedical Engineering and Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston Salem, NC, USA. To quantify minute forces with sub-pN precision on single molecules translocating through nanopores we developed real-time video-based 3D force detection for an optical tweezers system. With this setup, we investigate the threading force on a single dsDNA molecule inside silicon-nitride nanopores, as well as lipid-coated solid-state nanopores. We observe a strong dependence of the threading force on the nanopore size that can be related to the magnitude of the electro osmotic flow (EOF) inside the nanopore, which opposes the electrophoretic force. We show that the EOF can be additionally reduced by coating the nanopore wall with an electrically-neutral lipid bilayer, resulting in an 85% increase in threading force. Our experimental findings can be described by a quantitative theoretical model that incorporates a hydrodynamic slip effect by introducing a finite slip length of 0.5 nm at the dsDNA surface. 2549-Plat Adaptive Hopping Probe Ion Conductance Microscopy of Live Cells at ~5-10 NM Resolution A. Catalina Velez-Ortega1, Oleg Belov2, Pavel Novak3, Samir A. Rawashdeh4, Yuri E. Korchev5, Gregory I. Frolenkov1. 1 University of Kentucky, Lexington, KY, USA, 2Research Center for Audiology, Moscow, Russian Federation, 3Queen Mary, University of
517a
London, London, United Kingdom, 4University of Michigan - Dearborn, Dearborn, MI, USA, 5Imperial College London, London, United Kingdom. The inherent low imaging speed of hopping probe ion conductance microscopy (HPICM) poses a constraint for the study of living cells at super resolution. The duration of each imaging frame further increases when the cell topography requires large approach/retraction movements of the scanning pipette. We have modified our HPICM system to increase its imaging speed and sensitivity. We replaced the Z-scanner with a ~18 kHz resonant frequency piezo assembly to allow for faster pipette movements. An external proportional-integralderivative controller was used to decrease the delay in the Z-scanner. In our modified HPICM algorithm the approach speed adapts to the changes in the ionic current flowing through the pipette. Altogether, our changes to the HPICM system allowed for approach speeds up to 4X faster with delays of only ~50ms. Our ‘‘adaptive’’ approach curve effectively reduced the noise floor by half, which improved the vertical resolution of the system to ~5nm. We also introduced adaptive filtering of the ionic current, which decreased by half the minimum setpoint, thus improving the system’s sensitivity. To test the performance of the improved HPICM setup, we imaged the vertically protruding stereocilia (~0.5-3mm in height, ~100-400nm in diameter) of the rat inner ear sensory cells. When we previously imaged these structures with HPICM, we used glutaraldehyde-fixed cells and each imaging frame took >44 min (Novak et al. Nat Methods, 2009). Now, the entire stereocilia bundle (~9x9mm) in a live cell is imaged in ~15 min and sub-regions of interest (~2x2mm) in ~3-6 min, with ~10-30nm XY resolution, even with the hop amplitudes of 3-5mm. Our improved HPICM system allows for the faster imaging of live cells at nanometer resolution, even in the cells with very convoluted topographies. Supported by NIDCD/NIH (R01DC008861, R01DC014658). 2550-Plat Direct Observation of Transition Paths during the Folding of Proteins and Nucleic Acids Krishna Neupane1, Daniel A.N. Foster1, Derek R. Dee1, Hao Yu1, Feng Wang2, Michael T. Woodside1,2. 1 University of Alberta, Edmonton, AB, Canada, 2National Institute for Nanotechnology, Edmonton, AB, Canada. Transition paths are the trajectories followed during the fleeting moments when molecular structure changes during folding reactions. Because they provide a direct look at the intermediate transition states that dominate the dynamics of folding, transition paths encapsulate the critical information about how structure forms. Owing to their brevity, however, it has not previously been possible to measure transition paths directly, and only properties such as the average time to cross the transition path have been accessible. Using high-resolution optical tweezers to unfold and refold single molecules under mechanical load, we measured thousands of transition paths directly for both nucleic acid and protein folding, observing a great diversity of behavior as the molecules traversed the barrier region. Studying the distribution of transit times as a probe of the statistical nature of protein folding, we found that the transit times were broadly distributed, displaying an exponential tail. The average value and exponential decay of the distribution both agreed well with theoretical expectations for diffusion over the one-dimensional energy landscapes reconstructed for the same molecules by force spectroscopy, validating the fundamental physical picture of folding. These measurements provide a first look at the critical microscopic events that occur during structure formation in biomolecules, opening exciting new avenues for investigating folding phenomena. 2551-Plat Equilibrium Folding of an Individual Bacteriorhodopsin into and out of its Native Lipid Bilayer Resolves Energy Landscapes and Hidden Dynamics Hao Yu1, Matthew G.W. Siewny1, Devin T. Edwards1, Aric W. Sanders2, Thomas T. Perkins1,3. 1 JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, USA, 2Quantum Electronics and Photonics Division, National Institute of Standard and Technology, Boulder, CO, USA, 3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA. In protein folding, the complete characterization of all a protein’s folding intermediates and their dynamics is an essential prerequisite to developing an accurate model. An over-simplified view of folding processes emerges if briefly populated states and their dynamics remain undetected due to ensemble averaging and/or limited instrumental precision. We studied the folding of individual bacteriorhodopsins embed in their native lipid bilayer using cantilevers optimized for force spectroscopy with ~1-ms resolution. Improving temporal
