For measuring the minute forces exerted on single molecules during controlled translocation through nanopores with sub-piconewton precision, we have developed a video-based axial force detection and analysis system for optical tweezers. Since our detection system is equipped with a standard and versatile CCD video camera with a limited bandwidth offering operation at moderate light illumination with minimal sample heating, we integrated Allan variance analysis for trap stiffness calibration. Upon manipulating a microbead in the vicinity of a weakly reflecting surface with simultaneous axial force detection, interference effects have to be considered and minimized. We measured and analyzed the backscattering light properties of polystyrene and silica microbeads with different diameters and propose distinct and optimized experimental configurations (microbead material and diameter) for minimal light backscattering and virtually interference-free microbead position detection. As a proof of principle, we investigated the nanopore threading forces of a single dsDNA strand attached to a microbead with an overall force resolution of ±0.5 pN at a sample rate of 123 Hz.
Sebastian Knust¹, Andre Spiering¹, Henning Vieker², André Beyer², Armin Gölzhäuser², Katja Tönsing¹, Andy Sischka¹, and Dario Anselmetti¹: Rev. Sci. Instrum. 83, 103704 (2012)
¹: Experimental Biophysics & Applied Nanoscience, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany
²: Physics of Supramolecular Systems, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany
Copyright 2012 American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The article appeared in Review of Scientific Instruments 83, 103704 (2012) and may be found at http://link.aip.org/link/?RSI/83/103704.