Single beam optical tweezers setup with backscattered light detection for three-dimensional measurements on DNA and nanopores

Sischka, Andy; Kleimann, Christoph; Hachmann, Wiebke; Schäfer, Marcus; Seuffert, Ina; Tönsing, Katja; Anselmetti, Dario

doi:10.1063/1.2938401

Cited by 27 publications

(29 citation statements)

References 34 publications

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“…Finally, motions of the sample chamber relative to the microscope objective will induce a focal shift that displaces the trap axial position (Neuman et al, 2005). Recent efforts aimed at addressing some of these limitations include the unzipping of a known DNA template to obtain a calibrated reference (Deufel and Wang, 2006) or performing back-scattered light detection (optionally, with spatial filtering) to reduce systematic errors (Carter et al, 2007; Sischka et al, 2008). …”

Section: Instrument Calibrationmentioning

confidence: 99%

An Optical Apparatus for Rotation and Trapping

Gutiérrez–Medina

Andreasson

Greenleaf

et al. 2010

Methods in Enzymology

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We present details of the design, construction and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles (an 'optical torque wrench'). The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator (EOM), which affords improved performance over previous designs. The torque imparted to the trapped particle is assessed by measuring the difference between left-and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop. The limited angular range of the EOM (±180°) is extended by rapidly reversing the polarization once a threshold angle is reached, enabling the torque clamp to function over unlimited, continuous rotations at high bandwidth. In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces (~0.1-100 pN) and torques (~1-10,000 pN nm) in the study of biomolecules. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.

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Section: Instrument Calibrationmentioning

confidence: 99%

An Optical Apparatus for Rotation and Trapping

Gutiérrez–Medina

Andreasson

Greenleaf

et al. 2010

Methods in Enzymology

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“…The somewhat counterintuitive result is that the larger the nanopore the smaller the electrophoretic force becomes. The optical tweezersnanopore combination was demonstrated by several other laboratories [87][88][89], extended to DNA-protein complexes [90] and single RNA molecules [91]. Recent results combining nanocapillaries with optical tweezers [26,27] extend the range of possible applications and will facilitate incorporation of other optical techniques, such as single molecule fluorescence detection.…”

Section: Mechanical Controlmentioning

confidence: 99%

Controlling molecular transport through nanopores

Keyser

2011

J. R. Soc. Interface.

173

191

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Nanopores are emerging as powerful tools for the detection and identification of macromolecules in aqueous solution. In this review, we discuss the recent development of active and passive controls over molecular transport through nanopores with emphasis on biosensing applications. We give an overview of the solutions developed to enhance the sensitivity and specificity of the resistive-pulse technique based on biological and solid-state nanopores.

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“…Force measurement in z-direction basically requires an intensity detection of the forward or backscattered light coming from the trapped object. [6][7][8] To collect the forward scattered light, a condenser objective needs to be confocally adjusted to the trapping objective which limits the space between the two lenses and reduces the versatility of the setup. 9 To overcome this limitation, backscattered light detection can be utilized; 6,7 however, when operating this system in the vicinity of optical interfaces, disturbing force interference effects occur that can only be partially suppressed with an improved optical setup.…”

Section: Introductionmentioning

confidence: 99%

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

Knust

Spiering

Vieker

et al. 2012

Review of Scientific Instruments

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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.

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Single beam optical tweezers setup with backscattered light detection for three-dimensional measurements on DNA and nanopores

Cited by 27 publications

References 34 publications

An Optical Apparatus for Rotation and Trapping

An Optical Apparatus for Rotation and Trapping

Controlling molecular transport through nanopores

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

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