18-12-2012, 01:40 PM
Optical TWEEZERS
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Introduction:
The detection of optical scattering and gradient forces on micrometer sized particles was first reported in 1970 by Arthur Ashkin a scientist working at Bell Labs. Years later, Ashkin and colleagues reported the first observation of what is now commonly referred to as an optical trap: a tightly focused beam of light capable of holding microscopic particles stable in three dimensions.
One of the authors of this seminal 1986 paper, Steven Chu, would go on to use optical tweezing in his work on cooling and trapping neutral atoms. This research earned Chu the 1997 Nobel Prize in Physics.In an interview, Steven Chu described how Ashkin had first envisioned optical tweezing as a method for trapping atoms. Ashkin was able to trap larger particles (10 to 10,000 nanometers in diameter) but it fell to Chu to extend these techniques to the trapping of neutral atoms (0.1 nanometers in diameter) utilizing resonant laser light and a magnetic gradient trap (cf. Magneto-optical trap).
In the late 1980s, Arthur Ashkin and his colleagues first applied the technology to the biological sciences, using it to trap an individual tobacco mosaic vi-rus andEscherichia coli bacterium. Throughout the 1990s and afterwards, researchers like Carlos Busta-mante, James Spudich, and Steven Block pioneered the use of optical trap force spectroscopy to charac-terize molecular-scale biological motors. These molecular motors are ubiquitous in biology, and are responsible for locomotion and mechanical action within the cell. Optical traps allowed these biophysicists to observe the forces and dynam-ics of nanoscale motors at the single-molecule level; optical trap force-spectroscopy has since led to great-er understanding of the stochastic nature of these force-generating molecules
Optical Tweezers:
An optical tweezers is a scientific instrument that uses a focused laser beam to provide an attractive or repulsive force (typically on the order of piconew-tons), depending on the refractive index mismatch to physically hold and move microscop-ic dielectric objects. Optical tweezers have been par-ticularly successful in studying a variety of biological systems in recent years.
General Description:
Optical tweezers are capable of manipulating nano-meter and micrometer-sized dielectric particles by exerting extremely small forces via a highly fo-cused laser beam. The beam is typically focused by sending it through amicroscope objective. The nar-rowest point of the focused beam, known as the beam waist, contains a very strong electric field gradient. It turns out that dielectric particles are attracted along the gradient to the region of strongest electric field, which is the center of the beam. The laser light also tends to apply a force on particles in the beam along the direction of beam propagation. It is easy to understand why if one considers light to be a group of particles, each impinging on the tiny dielectric particle in its path. This is known as the scattering force and results in the particle being displaced slightly downstream from the exact position of the beam waist, as seen in the figure.
The electric dipole approximation:
In cases where the diameter of a trapped particle is significantly smaller than the wavelength of light, the conditions for Rayleigh scattering are satisfied and the particle can be treated as a point dipole in an inhomogenous electromagnetic field. The force applied on a single charge in an electromagnetic field is known as the Lorentz force.
EXPERIMENTAL DESIGN, CONSTRUCTION AND OPERATION:
Optical Tweezers use light to manipulate microscopic objects as small as a single atom. The radiation pres-sure from a focused laser beam is able to trap small particles. In the biological sciences, these instruments have been used to apply forces in the pN-range and to measure displacements in the nm range of objects ranging in size from 10 nm to over 100 mm.
The most basic form of an optical trap is diagramed in Fig 1a. A laser beam is focused by a high-quality microscope objective to a spot in the specimen plane. This spot creates an "optical trap" which is able to hold a small particle at its center.
Modern Optical Tweezers:
In practice, optical tweezers are very expensive, cus-tom-built instruments. These instruments usually start with a commercial optical microscope but add extensive modifications. In addition, the capability to couple multiple lasers into the microscope poses another challenge. High power infrared laser beams are often used to achieve high trapping stiffness with minimal photo-damage to biological samples. Precise steering of the optical trap is accomplished with lenses, mirrors, and acousto/electro-optical devices that can be controlled via computer. The most basic optical tweezers setup will likely include the following components: a laser (usually Nd:YAG), a beam ex-pander, some optics used to steer the beam location in the sample plane, a microscope objec-tive and condenser to create the trap in the sample plane, a position detector (e.g. quadrant photodiode) to measure beam displacements and a microscope illumination source coupled to a CCD camera.
Position detection
Sensitive position detection lies at the heart of quan-titative optical trapping, since nanoscale measure-ments of both force and displacement rely on a well-calibrated system for determining position. Position tracking of irregularly shaped objects is feasible, but precise position and force calibration
are currently only practical with spherical objects. For this purpose, microscopic beads are either used alone, or attached to objects of interest as “handles,” to apply calibrated forces. The position detection schemes presented here were primarily developed to track microscopic silica or polystyrene beads. How-ever, the same techniques may be applied to rack other objects, such as bacterial cells.
Optical Trapping (Blocks D & E)
A Nd-YAG laser beam (2W maximum at 1064nm) is used to form the optical trap (Block D: pink light path). The position of the trap in the specimen plane is adjusted using a set of Acousto-optic Deflec-tors(not shown). For position detection, the forward-scattered light off the trapped bead is collected with a condenser lens and the condenser back-focal plane is imaged in a Quadrant-photo-detector (QPD). The QPD output is digitized and stored on a PC for off-line analysis or observed in real-time with a spectrum analyzer. During combined optical trapping/single molecule fluorescence imaging it is known that ab-sorption of a NIR photon during the excited state lifetime of certain cyanine dyes (Cy3 and Alexa-555) can cause irreversible photo-destruction. Since the excited state lifetime of Cy3 is ~nsec while relevant experimental time scales are ~msec or longer a straightforward way to circumvent this problem is alternating the excitation and trapping beams. How-ever, chopping at frequencies close to the viscous relaxation time of the bead can compromise the per-formance of the trap and increase position noise. In our setup the same RF signal from a Voltage-controlled Oscillator drives both the 532nm and 1064nm Acousto-optic Deflectors, while an RF switch alternates between the two.
Applications:
The implications of a device capable of non-invasive translational manipulation of particles in the nanometer to micrometer range have not been fully realized by the scientific community; however, a flood gate of biological applications of the optical tweezer has greatly accelerated research in the field.
• Laser Scapel.
• Kinetic Studies of DNA and other Nucleic Acids.
• DNA Injection and/or incorporation.
• Controlled Cell Fusion.
• Microsurgery and manipulation of cells in vivo.
• Studies of in vitro Fertilization.
• Force measurements of Kinesin and other molecular motors.
• Mechanical Studies of Bacterial Flagella.
• Chromosome Dissection.
• Chromosome Manipulation during Mitosis.
• Gravity Perception in Plants.