Video-based and interference-free axial force detection and analysis for optical tweezers

Abstract

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.

Hydrodynamic Slip on DNA Observed by Optical Tweezers-Controlled Translocation Experiments with Solid-State and Lipid-Coated Nanopores

“…Then, optical tweezers were used to navigate the bead-DNA construct into close proximity with the NP (Figure A) and a membrane voltage of +50 mV was applied across the pore. The force threading the DNA into the NP and acting on the trapped bead was measured via our video-based force detection setup …”

“…The electrostatic component of the total force acting on dsDNA inside the NP can be estimated to be 1 pN/mV, however all DNA threading experiments yield a significantly lower effective threading force. ,,,, This is due to the hydrodynamic coupling of the EOF pointing in the opposite direction of the electrostatic force and therefore leading to a strongly reduced measured threading force . The EOF itself has its origin in the movement of the thin layer of counterions surounding the charged DNA backbone.…”

“…Afterwards, we introduced to the bottom compartment of the sample chamber NP-buffer solution containing 3.05 µm polystyrene microbeads (Spherotech, IL) conjugated with λ-DNA molecules (48.8 kbp, Roche Diagnostics, Germany) 15 . The force threading the DNA into the NP and acting on the trapped bead was measured via our video-based force detection setup 16 . The force threading the DNA into the NP and acting on the trapped bead was measured via our video-based force detection setup 16 .…”

“…The force threading the DNA into the NP and acting on the trapped bead was measured via our video-based force detection setup. 16 Coating of the NP walls with a lipid bilayer from small unilamellar vesicles (SUVs) and confirmation of bilayer stability was performed as detailed in ref 14. In brief, the SUVs are prepared by using a rotary evaporator to form a lipid film in a round-bottom glass flask.…”

Combining force and fluorescence microscopy for the manipulation and detection of single cells, viruses, and proteins

“…To conrm that with the use of a water immersion objective the shift in focus is indeed eliminated, we made use of the periodicity of the interference signal [83], [84]. To obtain the local periodicity of the interference signal, we splitted the traces as shown in Figure 4.27(a) in segments of 1110nm (see equation 3.4), and tted those with a sinusoidal function.…”