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TECHNIQUES
Optical trapping
Joshua Shaevitz
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Overview
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The basics
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What is it?
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Optical trapping is a technique that uses
light to position microscopic objects. Forces that result when an
intense beam of light strikes a small object trap it within the beam,
allowing the beam to "hold" the particle. In the biological sciences,
optical traps — also known as optical tweezers because they are used to
manipulate small objects — have been used to apply forces in the
pN-range and to measure nanometer-scale movements of objects ranging in
size from 10 nm to over 100 μm.
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How does it work?
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Figure 1
An optical trap is typically made by modifying a
conventional inverted microscope. A laser beam is focused by the
objective lens to a diffraction-limited spot above the specimen plane
where micron-sized objects become trapped.
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Figure 2
The trapped object interacts with the laser light by
bending and reflecting it. Because light carries momentum this
refraction of the laser beam imparts a force onto the object. The
reflected light creates a scattering force, while the refracted light
imparts the gradient force. The net result is a three-dimensional
spring which holds the bead in the focus of the laser beam.
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The most basic form of an optical trap is diagrammed in Figure 1.
A high-quality objective lens within a microscope is used to focus a
laser beam to a spot in the specimen plane. This spot creates an
"optical trap" which is able to hold a small particle at its center.
The forces felt by a particle within the trap consist of the light
scattering and gradient forces due to the interaction of the particle
with the light (Figure 2; see Details and variations).
Most frequently, optical tweezers are built by modifying a standard
optical microscope. These instruments have evolved from simple tools to
manipulate micron-sized objects to sophisticated devices under computer
control that can measure displacements and forces with high precision
and accuracy.
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Applications
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Optical tweezers have been used to trap
viruses, bacteria, living cells, organelles, small metal particles,
dielectric (electrically nonconducting) spheres, and even strands of
DNA. Applications include confinement and organization (e.g., for cell
sorting), tracking of movement (e.g., of bacteria), application and
measurement of small forces, and altering of larger structures (such as
cell membranes). Two of the main uses for optical traps have been the
study of molecular motors and the physical properties of DNA. In both
areas, a biological specimen is biochemically attached to a
micron-sized glass or polystyrene bead that is then trapped. By
attaching a single molecule of a molecular motor (such as kinesin,
myosin, RNA polymerase, etc.) to such a bead, it has been possible to
probe motor properties, such as: Does the motor take individual steps?
What is the step size? How much force can the motor produce? Similarly,
by attaching the beads to the ends of single pieces of DNA, experiments
have measured the elasticity of the DNA and the forces under which the
DNA breaks or undergoes a phase transition, and have even followed the
movement of a single polymerase molecule at near base-pair resolution.
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Details and variations
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Principle of operation. Figure 2
shows a more detailed look at how an optical trap works. The basic
principle behind optical tweezers is the momentum transfer associated
with bending light. Light carries momentum that is proportional to its
energy and in the direction of propagation. Any change in the direction
of light, by reflection or refraction, will result in a change of the
momentum of the light. If an object bends the light, changing its
momentum, conservation of momentum requires that the object must
undergo an equal and opposite momentum change. This gives rise to a
force acting on the object.
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In a typical optical tweezers setup, the
incoming light comes from a laser which has a "Gaussian intensity
profile." Basically, the light at the center of the beam is brighter
than the light at the edges. When this light interacts with a bead, the
light rays are bent according the laws of reflection and refraction
(two example rays are shown in Figure 2. The sum of the forces from
all such rays can be split into two components: Fscattering,
the scattering force, pointing in the direction of the incident light (z)
(see axes in Figure 2), and Fgradient,
the gradient force, which arises from the fact that the intensity of
the beam is greatest at its center and decreases toward its edges. The
gradient force is in the x-y plane and points toward the center of the
beam (dotted line). It thus acts as a restoring force that pulls the
bead toward the center of the trap. If the contribution to Fscattering
of the refracted rays is larger than that of the reflected rays, then a restoring force is also
created along the z axis, and a stable trap will exist at the position where all forces in the z
direction are balanced. The image of the bead can be projected onto a
quadrant photodiode to measure nm-scale displacements from the center
of the trap.
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When the bead is displaced from the
center of the trap, what force does it feel? The restoring force of the
optical trap works like an optical spring: the force is proportional to
the displacement from the center of the trap. In practice, the bead is
constantly moving with Brownian motion. But whenever it leaves the
center of the optical trap, the restoring force pulls it back to the
center. If some external object, like a molecular motor, were to pull
the bead away from the center of the trap, a restoring force would be
imparted to the bead and would oppose the force exerted by the motor.
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Figure 3
In order to follow the stepping of a molecular motor, a
motor-coated bead is held near the motor substrate (in this case a
kinesin and microtubule) with the optical trap. As the motor moves
along the substrate, it pulls the bead away from the center of the
optical trap resulting in a force on the motor.
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An example of an experiment involving an optical trap. Following
the movement of an individual molecular motor illustrates the uses and
capabilities of optical trapping. A good example is kinesin, a motor
that moves along the surface of microtubules. The basic setup for
following kinesin as it moves along a microtubule is shown in Figure 3.
In a typical assay of kinesin motion, purified kinesin molecules are
attached to the surface of small (~500 nm) glass beads. A variety of
methods and tests can be used to ensure that most beads have only one
kinesin molecule attached to them. The bead serves both as a handle
with which to position the kinesin molecule using an optical trap, and
as a way of knowing where the kinesin molecule is, since kinesin itself
is much too small to see. Microtubules adsorbed onto the surface of a
glass coverslip are used as tracks for the kinesin. While a bead is
viewed through an optical microscope, an optical trap is used to
capture it and move it into position above a microtubule. The trap is
used to keep the bead near the microtubule until the kinesin can bind
and the bead begins moving along it.
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Figure 4
As kinesin pulls the beam away from the optical trap
center, the laser beam direction can be used to detect the position of
the bead. When the kinesin steps, the optical trap is moved such that a
constant distance is maintained between the bead and optical trap,
insuring a constant force. A plot of kinesin position against time
showing five steps is displayed.
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In the absence of the trap, Brownian
motion of the bead — it would appear to be jumping all over the place —
would make it impossible to follow the bead's position with sufficient
accuracy to determine if kinesin moves continuously along the
microtubule or in a series of discrete steps, or to determine the size
of steps if they occur. With an optical trap, however, this can be
done. When the kinesin molecule first interacts with the microtubule,
the bead is in the center of the trap. As the kinesin molecule moves,
the bead is pulled away from the center, so that it experiences a force
in the direction opposite the one in which the kinesin is pulling it.
Being pulled in opposite directions considerably dampens the Brownian
vibrations of the bead and allows its position to be determined with an
accuracy of about 1 nm. (For comparison, the diameter of a microtubule
is 25 nm, and tubulin subunits repeat every 8 nm along its length.) By
adjusting the ATP concentration and using equipment that can record the
position of the bead many times a second, it becomes clear that kinesin
takes discrete steps of constant size along a microtubule, and that a
single molecule of kinesin can take multiple steps along a microtubule
before falling off. An example record of a single kinesin motor
exhibiting 8 nm steps against a 5-pN force is shown in Figure 4.
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Here a complication arises that
illustrates how many current experiments employ sophisticated
modifications beyond the basic concept of the optical trap. As a
kinesin molecule takes more and more steps, the bead is pulled farther
and farther from the center of the trap. With every step taken, the
bead experiences a stronger restoring force, and at some point the
restoring force is as great as that exerted on the bead by kinesin. At
this point the kinesin — and the bead — can move no farther.
Unfortunately, this occurs after only a few steps and the properties of
kinesin change as the force opposing it (the load) increases. As a
result, the basic experiment described above cannot give a picture of
how kinesin behaves under a constant set of conditions — as it would
when pulling a vesicle within a cell, for example — and how far it can
move along a microtubule. To overcome this problem and allow kinesin to
be observed as it operates under a constant opposing force, a very
fast, computerized feedback system is used to follow the position of
the bead and automatically move the trap along behind it. The system
maintains the bead at a constant distance from the center of the trap
so that the force exerted on it remains the same over long distances.
The force opposing the kinesin molecule is thus constant as it moves.
Under these conditions, it is clear that a single kinesin molecule is
capable of taking hundreds of steps of 8 nm along a microtubule.
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Modern optical tweezers. In practice, optical
tweezers are very expensive, custom-built instruments. These
instruments usually start with a commercial optical microscope but add
extensive modifications. High-power infrared laser beams are often used
to ensure that the restoring force of the trap increases sharply with
distance from its center — called high trapping stiffness. The use of
infrared beams also minimizes photo damage to biological samples.
Precise positioning of the optical trap is accomplished with lenses,
mirrors, and acousto/electro-optical devices that can be controlled via
computer. In addition, multiple lasers are often used so that more than
one object can be held simultaneously. In short, these are very
complicated instruments that require a working knowledge of microscopy,
optics, and laser techniques.
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Contributed by
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Joshua Shaevitz
University of California, Berkeley
Department of Integrative Biology
3060 Valley Life Sciences
Berkeley, CA 94720-3140
E-mail: jshaevitz@berkeley.edu
Last Revised on April 20, 2005
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©Jones and Bartlett Publishers (2007)
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