Force determination in lateral magnetic tweezers combined with TIRF microscopy

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA:protein interactions. In particular, combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of the flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.We thank the financial support from the Spanish MINECO (FIS2014-58328-P) and from (ERC) under the European Union’s Horizon2020 Research and Innovation programme (grant agreement No 681299). J. M. M. acknowledges a Predoctoral PhD fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174). ). G. L. M. F was supported by the Biotechnology and Biological Sciences Research Council (1363883). M. S. D was supported by the Wellcome Trust (100401 and 077368).Peer reviewe

Gold nanoparticle coated silicon tips for Kelvin probe force microscopy in air

Abstract The tip apex dimensions and geometry of the conductive probe remain the major limitation to the resolution of Kelvin probe force microscopy (KPFM). One of the possible strategies to improve the spatial resolution of surface potential images consists in the development of thinner and more durable conductive tips. In an effort to improve the lateral resolution of topography and surface potential maps, we have evaluated high aspect ratio conductive tips created by depositing gold nanoparticles on standard silicon tips. Besides the already known general topographic resolution enhancement offered by these modified tips References [1] L