英文介紹Introduction

Our Optical Tweezers System Provides:

• Quantitative 3D Force Measurements with 0.1 pN Resolution

• Achievable Trapping Force of 400 pN with 1 W Fiber Laser

• Manipulation of Trapped Objects with Nanometer Precision

• Compact and Ultrastable Modular System

• Programmable LabView? Software Interface

• Easy-to-use Force Calibration without Detector Alignment

• Scope of Applications:

  • from Single Molecules to Living Cells

  • from Polymer Elasticity to Microfluidics

  • from Molecular Interactions to Nanopores

  
   

Optical Trapping and Force Measurement

Optical tweezers are used to trap and actively manipulate microscopic objects. They also offer a vast area of applications by measuring forces applied to trapped objects.

The Optical Trap

Microscopic objects - like individual nano- or microparticles, cells, cell compartments, single or clustered molecules - can be trapped securely inside the center of a strongly focused laser beam.

When an external forces is acting on the trapped object, it deflects from the center of the trap as the deflection x depends linearly on trap stiffness k and force F.

Forces

A trapped particle experiences various external forces. Atoms or molecules of the surrounding medium induce Brownian motion in all three dimensions, depending on temperature, viscosity and the presence of obstacles in the vicinity. Macroscopic fluid movements cause a drag force. Electric fields and bulk or surface charges may generate electrophoretic or electroosmotic forces.

Particularly, single molecules can induce forces of broad variety and magnitude while bound to the trapped object. On the other hand, the application of a force generated by an optical trap to a single molecule will gain vast insight into the molecular structure and elasticity, binding properties and kinetics.

Deflection is the Essence

Generating and metering various forces requires a reliable force measurement capability in all three dimensions to allow for a maximum degree of experimental freedom and versatility. Therefore, force detection is accomplished by precisely measuring the deflection of the trapped particle in each direction.

The PicoTweezers system utilizes a sophisticated and easy-to-use video analysis (right image below) for particle tracking, detection and force measurements. It offers the largest field of application since it clears common calibration difficulties, system instabilities, as well as experimental and spatial restrictions.

Video Detection and Analysis

Video-based force detection is easy to calibrate and provides an alignment-free and unsusceptible method for all force measurements in three dimensions. It is embedded into the LabView? platform.

The Principle

In addition to a video camera imaging the surrounding area of the optical trap, a second high-speed CMOS camera simultaneously surveys the magnified image of the trapped particle. The software searches for specific edges (as shown in the upper right quadrant of the image) in each frame and in real-time, determines gradients (lower right quadrant) and fits a circle (lower left quadrant) which correlates to the apparent particle diameter.

If an external axial force is acting on the particle, its apparent diameter changes, which the software translates into a z-force. Lateral forces only shift the center of the particle. These lateral deflections in the order of nanometers are then translated into x- and y-forces.

Easy and Reliable Force Calibration

Force calibration in three dimensions is conducted by moving the surrounding medium via the piezo stage using Stokes’ drag force law.

For specific applications, video-based force detection utilizes Allan Variance analysis and calibration. Here, smallest particle fluctuations are recorded and analyzed without the need of applying any frictional force. Both Stokes’ method and Allan Variance do not require the determination of the trap stiffness k, though the video-analysis software can calculate it if desired.

Benefits of Video-Based Force Detection

There is no need of detector alignment or adjustment in the beginning or during experimentation because the CMOS camera providing data for video detection and analysis is integrated into the optical pathway between laser and optical trap. Video detection is unsusceptible to disturbing particles that occasionally may be trapped together with the measured object. Since the diameter of the trapped object is permanently monitored, further particles of interest can be trapped and compared with previous ones. Specifically tailored Allan Variance for video analysis is a powerful calibration tool for experiments that take place in an environment that prevents other calibration or analysis methods. When trapping particles close to interfaces (bottom or ceiling of sample chamber, artificial or biological membranes, etc.), video analysis delivers an interference-free force signal.

Detection Tandem

Optionally, PicoTweezers can be equipped with additional backscattered light detection capability for simultaneous measurements or as stand-alone method, if experiments need to be conducted in absence of light or if particle fluctuations must be analyzed with highest sample rate in the kHz range.

Applications — Single Molecules and Polymer Elasticity

The elastic behavior of a single DNA-strand in absence or in presence of binding ligands can be reliably measured. Theoretical polymer models that are fitted to the results will deliver parameters, which characterize the polymer elasticity.

Grabbing a Single Molecule

To bind a single DNA-strand between two coated microbeads, it has to be properly functionalized on both ends. Thus, it can be immobilized between two beads, of which one is optically trapped and the other is held on the tip of a micropipette. Increasing the distance between the beads by moving the piezo stage induces a controlled mechanical tension to the DNA.

As a response, the force-extension curve of the molecule exhibit characteristic mechanical properties, such as an entropic elasticity, an overstretching plateau and a melting transition region.

DNA as Sensor for Foreign Molecules

The DNA strand can serve as a host for a variety of different molecules, such as small intercalators, groove-binders, proteins, enzymes or molecular motors.

The binding event of a single or a multitude of ligands can change the elastic response more or less significantly. As an example, the force curve of a DNA is shown in presence of the antibiotic distamycin-A that attaches to the minor groove of the DNA strand while stabilizing it and helping to resist the overstretching. On the other hand, diazoniapentaphene as an intercalator increases both contour and persistence length and renders the overstretching plateau to disappear.

Applications — Translocation through Nanopores

Nanopores play a major role in biology and they rapidly evolved into a new and promising technique in single-molecule detection. The controlled threading of a single DNA molecule or a DNA-protein complex into a nanopore allows investigation of the translocation dynamics and a localization of the bound protein.

Translocating a Single DNA Strand

When the DNA on the trapped bead approaches the nanopore (to a distance of 5 μm) it is immediately threaded into the pore by electrostatic forces acting on the negatively charged DNA backbone. This effect can be monitored as an abrupt step of the force signal to a certain value, which remains constant even when retracting the bead. The measured force depends on the applied voltage, as well as on the diameter of the nanopore.

When the entire DNA strand with an end-end-distance of 10.5 μm is pulled out of the pore, the force drops back to zero.

Single DNA-Bound Protein

A distinct asymmetric force signal occurs when a single peroxiredoxin molecule bound to the DNA stand is actively pulled through the pore. This effect serves as a label-free localization of the protein binding site.

It can be understand as the result of an effective positive charge of the protein counteracting the negative DNA backbone charge and reducing the electrostatic force.

We Design and Build Your Optical Tweezers.

PicoTweezers is a stand-alone system, that can also be customized to your Zeiss Axiovert, Axio Observer A1 or D1. It will be equipped with a 1 W or 5 W IR fiber laser for highest spatial trap stability yielding a trapping force of at least 400 pN or 2 nN, respectively.

The 3D-piezo stage enables nanometer resolution in a range of 200 μm in x and y, as well as 20 μm in z-direction. Video-analysis can achieve a lateral and axial resolution of at least 2.5 nm, which results in a force resolution of 0.1 pN with a frame rate of 200 and 400 Hz in z- and x,y-direction, respectively

References

• S. Knust, A. Spiering, H. Vieker, A. Beyer, A. G?lzh?user, K. T?nsing, A. Sischka and D. Anselmetti
  Video-Based and Interference-Free Axial Force Detection and Analysis for Optical Tweezers
  Review of Scientific Instruments, 83, 103704 (2012)

• A. Spiering, S. Getfert, A. Sischka, P. Reimann and D. Anselmetti
  Nanopore Translocation Dynamics of a Single DNA-Bound Protein
  Nano Letters, 11, 2978 (2011)

• A. Sischka, A. Spiering, M. Khaksar, M. Laxa, J. K?nig, K.J. Dietz and D. Anselmetti
  Dynamic Translocation of Ligand-Complexed DNA Through Solid-State Nanopores with Optical Tweezers
  Journal of Physics - Condensed Matter, 22, 454121 (2010)

• C. Kleimann, A. Sischka, A. Spiering, K. T?nsing, N. Sewald, U. Diedrichsen and D. Anselmetti
  Binding Kinetics of Bisintercalator Triostin A with Optical Tweezers Force Mechanics
  Biophysical Journal, 97, 2780 (2009)

• D. Pla, A. Sischka, F. Albericio, M. Alvarez, X. Fernandez-Busquets and D. Anselmetti
  Optical-Tweezers Study of Topoisomerase Inhibition
  Small, 5, 1269 (2009)

• A. Sischka, C. Kleimann, W. Hachmann, M.M. Sch?fer, I. Seuffert, K. T?nsing and D. Anselmetti
  Single Beam Optical Tweezers Setup with Backscattered Light Detection for Three-Dimensional Measurements on DNA and Nanopores
  Review of Scientific Instruments, 79, 063702 (2008)

• D. Anselmetti, N. Hansmeier, J. Kalinowski, J. Martini, T. Merkle, R. Palmisano, R. Ros, K. Schmied, A. Sischka and K. T?nsing
  Analysis of Subcellular Surface Structure, Function and Dynamics
  Analytical and Bioanalytical Chemistry, 387, 83 (2007)

• A. Sischka, K. T?nsing, R. Eckel, S.D. Wilking, N. Sewald, R. Ros and D. Anselmetti
  Molecular Mechanisms and Kinetics between DNA and DNA Binding Ligands
  Biophysical Journal, 88, 404 (2005)

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