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Laser Tweezers
Experimental Procedure
Ernest Kempton Adams Laboratory
The following pages are meant to briefly introduce you to the history of laser
tweezers, the theory behind them, and how to use our particular apparatus to make a few
of the many measurements possible with this device. They are meant as a primer to
optical trapping and as a guide to the idiosyncrasies of our particular setup. However, it is
not the intention that you will use this guide as your only source of information on this
interesting and useful application of laser light. An extensive list of references -- with
brief synopses where necessary -- are included in appendix A. In addition, many of these
references, including the book, Laser Tweezers In Cell Biology, ought to accompany the
apparatus.
Introduction
History
Staring through a microscope at Bell Labs in New Jersey, Dr. Art Ashkin sat
looking through his microscope onto the miniature world below. His sample, which had a
laser focused on it, was behaving in a way that he did not expect. To his surprise, he
noticed that some of the particles in his sample began to "stick" to the laser beam and
follow it around as the laser tracked across the field of view. After further investigation,
it was clear that for some reason the laser beam was in fact acting like a pair of
microscopic tweezers, squeezing particles at the point of its focus and holding them
stationary while the rest of the sample moved about it. This accidental discovery led to a
paper published by Ashkin's group in 1986, reporting the achievement of "the first
experimental observation... of a single-beam gradient force radiation pressure particle
trap." This serendipitous development would lead to an entirely new technique for
manipulating microscopic and sub-microscopic particles, eventually being applied to
individual atoms. Due to their ability to precisely manipulate microscopic particles, laser
tweezers lend themselves to a wide array of different applications in experimental
physics, biology and chemistry. Objects that are smaller than ten microns-a human hair is
100 microns thick-can be trapped and moved about. In addition, because the shape of the
potential energy well in which the particle is trapped can be well characterized,
measuring forces on the order of piconewtons is possible. To get a sense of the scale of a
piconewton, the force on a kilogram mass on the Earth’s surface is roughly 10 newtons,
while a piconewton is one trillionth of this. These two abilities, manipulation and force
measurement, can be used to accomplish many novel procedures.
One such procedure results from the accessibility of lasers to spaces that are
unreachable with solid instruments. Most devices that dexterously manipulate matter
require a solid handle to be attached to the grasping tip, a forklift or a set of forceps, for
example. Laser tweezers require only a transparent through which a laser beam can
travel. A potent illustration of this advantage is seen in the manipulation of organelles
within the confines of a living cell. Light from the laser passes through the cell wall and
only affects the organelle that it is focused on. In this way, properties of internal structure
of living organisms can be explored. Laser tweezers have also been used by scientists to
measure the forces on microscopic particles. Although many biological substances cannot
be trapped directly, they can be attached to particles that are easily trapped. A molecule
can be stretched out like springs between a solid anchor and the particle controlled by the
laser. The molecule's stiffness can be calculated by observing the distance out of the
center of the trap traveled by the entrapped particle when pulled out of the focal point of
the laser by another force, which could arise from either a second optical trap or the
attachment of part of the object to a fixed surface. The springiness of DNA and elastic
muscular proteins are measured in this way. Motile cells such as sperm and flagellants
can be tested to determine the strength of their swimming under different conditions. This
is noted by observing their ability to swim out of traps of varying strengths. Sperm are
subjected to varied temperatures and environmental chemistry in order to find the ideal
conditions for insemination. Related to reproduction, artificial insemination can be aided
by the placement of sperm cells in the proximity of an egg in order to increase the chance
of conception. Multiple lasers focused on the same sample can be used to manipulate
several different objects simultaneously. Two cells can be brought together and, after
their membranes are cut open, fused into a single hybrid cell. In addition, arrays of
tweezers can be used to create complex three-dimensional structures out of molecules
that can then be frozen into place. This technique is currently being developed and may
lead to the ability to mass-produce micro devices with useful electronic and mechanical
properties.
Current uses
Laser tweezers continue to be of great use in basic research. Due to advances in
diode laser technology, it is now possible to access powerful lasers in a variety of
wavelengths at lower costs than ever before. The laser tweezers is a very useful tool for
biophysicists who wish to measure the miniscule forces exerted by the cellular
interactions in vivo, an important area of current research. With the availability of a large
variety of commercially available protein coated polystyrene beads, researchers are able
to probe interactions between the cell and its external environment with precision using
this very flexible experimental technique. A search of current scientific journals will
yield a panoply of other current applications.
This setup
The purpose of the apparatus and this procedure is to familiarize you with the
basic theory and function of laser trapping. Though research grade laser traps are
generally much more powerful (and expensive), the apparatus you will be working with
is, delicate, costly, potentially dangerous, and can perform many of the same tasks as
more elaborate setups. Caution should be taken when working with this apparatus both
when the laser in on as well as when it is off. A slight perturbation to the optical path can
misalign the laser and require a tedious and time-consuming period of realignment. The
laser that traverses this optical path has a power of close to 30mW and is carried in the
form of 633 nm (red) photons that are readily absorbed by the retina. Thus, in the best
interest of your eyes, you must wear goggles when the primary laser is operating.
The moment you relax around the laser is the moment that an accident will happen, no
matter how comfortable you may feel.
Figure 1
Figure 2
Getting Familiar With the Apparatus
Optical Path
In order to familiarize you with the apparatus, start at the source of the optical
path, the laser itself. The laser uses He-Ne gas to produce a coherent emission that is
capable of a maximum output of 30mW at 632.8nm. The laser is attached to its power
supply that is turned on via a key.
The next elements in the optical path are two mirrors. These mirrors serve to
redirect the laser beam. These mirrors allow us to steer the laser beam at any angle and at
any height (within the confines of the range of much of the mirrors). The beam is steered
by these mirrors into a set of two lenses. These lenses act as a telescope, increasing the
diameter of the beam for reasons that will be explained later.
Figure 3
After the beam passes through the telescoping lenses, it is vertically redirected by
a third mirror. This mirror serves to send the beam through a third lens that will give the
beam front the optimal radius of curvature. The beam then strikes a fourth mirror and is
redirected into a side port that is bored into the side of the aluminum adapter sitting on
top of the microscope. The adapter holds two dichroic reflectors. These act as mirrors for
long wavelength light while being transparent for light of higher frequencies. Thus, when
the red laser beam meets the dichroic surface it reflects down the barrel of the microscope
where it then meets the back of the microscope objective.
The use of the lenses in the beam path previously discussed is entirely based on
the properties of the objective. The telescope is de-signed to create a beam diameter that
matches the diameter of the back of the objective. The result is that all of the laser light
enters the objective so that no power is lost and that the laser light entirely fills the back
of the objective, allowing the area of glass to be utilized in re-solving the smallest spot
possible. The focusing power of the objective is further maximized because the third lens
has created a beam front with a radius of curvature ideal for the objective.
The objective creates a highly focused spot that will act as our optical trap once a
suitable sample is placed on the microscope stage.
Of course, the microscope objective continues to focus light in the other direction
as well, sending it up the microscope barrel. This light once again passes through the first
dichroic reflector. Here the dichroic serves to weaken the intense laser beam so that our
detectors are not damaged. The image from the microscope continues upward through the
aluminum adapter, striking a second dichroic before reaching the camera. The reflected
laser light from this second dichroic will eventually exit the upper side port and be
absorbed by a photodiode array that will indicate small changes in a trapped particles
position.
Procedures
Rough Alignment of the Trap:
All coarse alignment is to be done with a secondary, less powerful laser source, or by
filtering out some of the intensity of the present laser.
1. Align laser path without lenses in place.
a.
Rotate microscope objective holder until there is no objective in the optical
path. If there are no empty holes, carefully unscrew one of the objectives from the
microscope.
b.
Open both apertures in the microscope condenser unit. Check that the
apertures are open by turning on microscope illumination and verifying the
maximum amount of light passes through condenser. Place a square of paper
beneath the condenser, resting on the foot of the microscope, covering the
illumination window.
c.
Roughly align mirrors by unfastening thumbscrews in post bases and rotating
the posts until the laser beam reflects off of each mirror down the optical path.
Align the dichroic assembly by unscrewing its fastening thumbscrew and rotating
the assembly. You should see laser light on the paper beneath the condenser.
d.
Using fine adjustment screws on mirror mounts, steer the path so that you
maximize the amount of laser light passing through the condenser and falling on
the paper. If necessary, repeat step c) such that the laser is reflected from the
centers of the mirrors in order to allow maximum adjustment flexibility in later
steps.
e.
Place spare posts identical to those of the lenses into the lens post holders.
Essentially you mimic adding the lenses, by adding lens posts without a lens.
f.
By adjusting the height of the lens posts, ensure that the laser beam passes
directly across the center of each post. This ensures that when you add the lenses
the laser beam can be made to travel directly through the center of each lens.
Repeat steps c), d), and f) as necessary.
g.
Now, close the condenser apertures all the way down. If the beam is precisely
aligned, you should still see the laser spot brightly.
h.
Finally, check that the beam is perpendicular to the stage by holding a blank
microscope slide flat on the stage in the path of the laser beam. By tilting the slide
slightly, you should be able to find the reflection on mirror 4. If, when the slide
lies flat on the stage the reflected beam strikes the mirror at the same spot as the
incident beam, the beam is perpendicular with the stage.
2. Add lenses
a.
Mark the position of the spot that your now well-aligned laser beam makes on
the paper below the condenser.
b.
Remove the bare post from lens 1 position and replace it with a post with the
appropriate lens attached.
c.
Adjust the lens position until the spot on the paper returns to the spot you
marked in 2a).
d.
Repeat steps 2b) and 2c) for lens 2 and lens 3.
3. Verify rough alignment through video system.
a.
Switch on monitor and video camera. Adjoin the camera to the system by
placing the camera’s lens cylinder into the cylindrical opening at the top of the
dichroic assembly.
b.
Turn on microscope illumination. Place a blank slide in microscope stage
slide holder. Using a low power objective, focus the microscope onto the top
surface of the slide. You may need to mark on the slide with a marker in order to
have a reflective surface onto which you can focus.
c.
You should see the reflection of the laser from the surface of the glass slide.
Verify that this reflection is from the laser by slightly adjusting the laser beam
using the fine adjustments on mirror 4.
Procedure for Preparing a Bead Sample:
1.
2.
3.
4.
5.
6.
7.
8.
Using a glass transfer pipette and a rubber bulb, measure out 10 ml of distilled
water into a graduated cylinder. For a very dilute sample, measure 15-30 ml of
distilled water. For a more concentrated sample of beads, use less distilled water.
From the refrigerator, take a dropper bottle containing the appropriately sized
beads.
Shake the dropper bottle until the beads enter a suspension. There should be
no clumps of beads and the suspension should resemble milk. If shaking the bottle
does not provide adequate agitation, you may need to remove the dropper bottle’s
cap and use a pipette to repeatedly take up and expel the contents of the bottle
until the suspension becomes homogeneous.
Squeeze a single drop of the bead suspension into the distilled water. Using
the pipette, take up and expel the distilled water until the new, dilute suspension
is thoroughly mixed.
Taking a clean cover slip and using the vacuum grease filled syringe, form a
thin but continuous border of grease along the edges of a cover slip on one side.
Lay the cover slip down, grease side facing up.
Using a transfer pipette, add enough of your diluted bead suspension to the
grease well on the cover slip, so that the suspension covers the bottom inside of
the well.
Place a clean microscope slide on top of the cover slip. This should be done in
such a way that the grease on the cover slip makes a seal against the slide but
does not seal until all the air inside is expelled. This is done in order to prevent
bubbles from being present in your sample.
Pick up your slide and flip it over. Using a wipe, dry any suspension that was
expelled from the well in step 7.
Procedure for Measuring the Maximum Trapping Strength:
In order to empirically determine the maximum strength of your laser trap you will
utilize an equation that gives a simple description of the viscous drag force that acts
between a bead held stationary by the laser tweezers and the surrounding fluid in your
sample. This equation, called the Stokes drag equation, is of the form:
F = 6 πηrv,
where η is the viscosity of the water, which is approximately 10-3 Ns/m2, v is the escape
velocity of the bead from the trap and r is the radius of the sphere.
The Stokes equation will not hold if the bead is less than several bead diameters from
the top of the cover slip due to surface effects that affect the viscous drag. In addition,
spherical aberrations will drastically reduce trapping strength with increasing depths.
Therefore, all measurements of the trapping strength should be done at a few
diameters below the surface.
Recording the Bead Escape Velocity:
1.
2.
3.
4.
5.
6.
Align the laser tweezers following the appropriate procedure.
Prepare a sample of beads following the appropriate procedure.
Place the slide, cover slip up, in the microscope sample holder.
Trap a single bead just below the cover slip surface.
Turn on the VCR system and begin recording.
Translate the stage with a slight acceleration until the bead falls out of the laser
trap.
7. Stop recording. Make a note of the tape position and record the diameter of the
beads used for this measurement.
8. Repeat steps 2-7 for differently sized beads.
Analyzing the Bead Escape Velocity:
1. Advance the tape to the point just before the bead escapes from the trap and,
using the VCR remote control, press the pause button.
2. Using the frame advance button of the VCR, measure the distance that an object
that is affixed to the cover glass (a stuck bead, a piece of dust, etc.) moves with
each depression of the advance button.
Calibrating Distance Scales:
Distance scales for the video system are easily found by noting the resulting image
size for a bead of known diameter.
Calibrating Time Scales:
Time elapsed with each depression of the frame advance is 1/30 of a second. This can
be verified by making a recording of an image that changes regularly with a known
period and than counting the number of frame advance depressions required to advance
through an entire period. Making a recording of the second hand of a watch is one such
example.