Extending calibration-free force measurements to optically-trapped rod-shaped samples.
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ABSTRACT:
SUBMITTER: Catala F
PROVIDER: S-EPMC5318951 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Magnetic tweezers are a wide-spread tool used to study the mechanics and the function of a large variety of biomolecules and biomolecular machines. This tool uses a magnetic particle and a strong magnetic field gradient to apply defined forces to the molecule of interest. Forces are typically quantified by analyzing the lateral fluctuations of the biomolecule-tethered particle in the direction perpendicular to the applied force. Since the magnetic field pins the anisotropy axis of the particle, the lateral fluctuations follow the geometry of a pendulum with a short pendulum length along and a long pendulum length perpendicular to the field lines. Typically, the short pendulum geometry is used for force calibration by power-spectral-density (PSD) analysis, because the movement of the bead in this direction can be approximated by a simple translational motion. Here, we provide a detailed analysis of the fluctuations according to the long pendulum geometry and show that for this direction, both the translational and the rotational motions of the particle have to be considered. We provide analytical formulas for the PSD of this coupled system that agree well with PSDs obtained in experiments and simulations and that finally allow a faithful quantification of the magnetic force for the long pendulum geometry. We furthermore demonstrate that this methodology allows the calibration of much larger forces than the short pendulum geometry in a tether-length-dependent manner. In addition, the accuracy of determination of the absolute force is improved. Our force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.
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Project description:Force measurement with an optical trap requires calibration of it. With a suitable detector, such as a position-sensitive detector (PSD), it is possible to calibrate the detector so that the force can be measured for arbitrary particles and arbitrary beams without further calibration; such a calibration can be called an "absolute calibration". Here, we present a simple method for the absolute calibration of a PSD. Very often, paired position and force measurements are required, and even if synchronous measurements are possible with the position and force detectors used, knowledge of the force-position curve for the particle in the trap can be highly beneficial. Therefore, we experimentally demonstrate methods for determining the force-position curve with and without synchronous force and position measurements, beyond the Hookean (linear) region of the trap. Unlike the absolute calibration of the force and position detectors, the force-position curve depends on the particle and the trapping beam, and needs to be determined in each individual case. We demonstrate the robustness of our absolute calibration by measuring optical forces on microspheres as commonly trapped in optical tweezers, and other particles such a birefringent vaterite microspheres, red blood cells, and a deformable "blob".
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Project description:Magnetic tweezers are a powerful technique to perform high-throughput and high-resolution force spectroscopy experiments at the single-molecule level. The camera-based detection of magnetic tweezers enables the observation of hundreds of magnetic beads in parallel, and therefore the characterization of the mechanochemical behavior of hundreds of nucleic acids and enzymes. However, magnetic tweezers experiments require an accurate force calibration to extract quantitative data, which is limited to low forces if the deleterious effect of the finite camera open shutter time (?sh) is not corrected. Here, we provide a simple method to perform correction-free force calibration for high-throughput magnetic tweezers at low image acquisition frequency (fac). By significantly reducing ?sh to at least 4-fold the characteristic times of the tethered magnetic bead, we accurately evaluated the variance of the magnetic bead position along the axis parallel to the magnetic field, estimating the force with a relative error of ~10% (standard deviation), being only limited by the bead-to-bead difference. We calibrated several magnets - magnetic beads configurations, covering a force range from ~50?fN to ~60?pN. In addition, for the presented configurations, we provide a table with the mathematical expressions that describe the force as a function of the magnets position.
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Project description:IntroductionOptical trapping is a laser-based method for probing the physiological and mechanical properties of cells in a noninvasive manner. As sperm motility is an important criterion for assessing the male fertility potential, this technique is used to study sperm cell motility behavior and rotational dynamics.Methods and patientsAn integrated optical system with near-infrared laser beam has been used to analyze rotational dynamics of live sperm cells from oligozoospermic and asthenozoospermic cases and compared with controls.ResultsThe linear, translational motion of the sperm is converted into rotational motion on being optically trapped, without causing any adverse effect on spermatozoa. The rotational speed of sperm cells from infertile men is observed to be significantly less as compared to controls.ConclusionsDistinguishing normal and abnormal sperm cells on the basis of beat frequency above 5.6 Hz may be an important step in modern reproductive biology to sort and select good quality spermatozoa. The application of laser-assisted technique in biology has the potential to be a valuable tool for assessment of sperm fertilization capacity for improving assisted reproductive technology.
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