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Construction of Optical Tweezers
(From Volume II, Section 7 of
Cells: A Laboratory Manual )
Steven M. Block,
Department of Molecular Biology Princeton University
E-mail:
[email protected]
This selected chapter represents a contribution to
Cells: A Laboratory Manual
(David Spector, Robert Goldman, Leslie Leinwand, eds.).
Prepared by Dr. Steven M. Block of Princeton University,
the chapter describes the construction of optical tweezers,
a single beam optical trap that uses highly focused laser
light to trap and manipulate microscopic biological objects,
including cells, organelles and chromosomes.
Chapter outline
- Introduction to Optical Trapping
- Laser Beam Steering
- Building the Trap
- List of Parts
- Putting it all Together
- Alignment Procedure
- Variations on a Theme:
How to "soup up" this design
- Selected References
- Figures
- Beam steering and conjugate planes
- Design for a simple Steerable
optical trap
CONSTRUCTION OF OPTICAL TWEEZERS
INTRODUCTION TO OPTICAL TRAPPING
A single-beam optical trap, or optical tweezers, uses highly
focused laser light to grab and manipulate microscopic objects
(for reviews on the principles and applications of optical trapping,
see Selected References at the end of the chapter). Optical tweezers
derives its unique trapping capability from the three-dimensional
gradient in light intensity found near a focus. When a moderately
powerful laser is focused to a diffraction-limited spot in the
specimen plane of a microscope, a steep gradient in light is produced
in the focal region. Small dielectric objects, such as latex or
silica micro-spheres, or biological material, including cells,
chromosomes, and organelles, experience a form of radiation pressure,
called the �gradient force�, that tends to draw them towards the
center of that region. Another form of radiation pressure, usually
called the scattering force, arises from reflection or absorption of
light, and it tends to push objects down along the beam, in the
direction of propagation of the light, much as a stream of water
from a fire hose propels objects away from the nozzle. Stable,
three-dimensional trapping takes place when the effect of gradient
force is sufficiently large to overcome the fire hose effect of the
scattering force. In practice, such a condition can be achieved by
using microscope objectives with the highest possible numerical
aperture, which generate the steepest gradients. The numerical
aperture (NA) equals the index of refraction of the immersion fluid
(air, water, or oil) multiplied by the sine of the half-angle of
opening of the focused light: typical values for high NA objectives
are in the range of 1.001.40, corresponding to full angles of
opening up to 140°.
In practice, trapping lasers focus from 10 mW to 1 W of light into
the microscope, producing huge fluxes at the specimen plane, ranging
from 106108 W/cm2. To diminish
the possibility of optical damage (opticution) arising from
these enormous power levels, a laser whose wavelength lies in the near
infrared region is generally used. This is because visible light is
heavily absorbed in naturally-occurring pigments found in biological
material, while far infrared light is absorbed by water. Most
biological specimensbut not allare fairly transparent in
between, over the near infrared region from 7001300 nm. Near
infrared sources with sufficient power to trap include the
Nd:YAG [neodymium: yttrium-aluminum-garnet] and
Nd:YLF [neodymium: yttrium-lithium-fluoride] lasers, at 1064
and 1047 nm, respectively, the Ti:Al2O3
[titanium: sapphire] laser, continuously tunable from 6951100 nm,
and various diode lasers, available at wavelengths from
7001300 nm, with highest powers from 800900 nm. All are
suitable for trapping, but vary widely in cost, ease of use, etc.
To produce the steepest light gradient, trapping lasers are run in
the lowest-order mode, called TEM00, or, for diode lasers,
single mode. Laser prices vary widely, depending on the power and
laser type. At lower power levels, diode lasers are the most
convenient choice, and, as with most developments of the semiconductor
industry, the average cost per unit is dropping and the average power
per unit is increasing. However, high power single mode diode lasers
must be corrected for their non-circular beams and astigmatism using
additional optics (cylindrical lenses or anamorphic prisms), and often
require thermoelectric cooling. Some laser diode manufacturers will
provide these items in a ready-to-go package, or you can do it yourself.
An optical trap based on a 200 mW diode laser (850 nm) is available
commercially (LaserTweezers® 2000, Cell Robotics, Albuquerque NM).
Nevertheless, for reasons of economics, practicality, modifiability,
or individuality, you may wish to roll your own. This chapter covers
the basic principles involved in building optical tweezers. The device
described here, while quite simple overall, produces a fully-functional
optical trap that can be steered in the specimen (x-y) plane as
well as in the axial (z-) direction using external optics. The
design features a shutter for safety, an attenuator for variable power
operation, and easy alignment (crucial!). Most of the parts are
standard components available from such optical manufacturers as
Ealing, Edmund, Melles Griot, Newport, New Focus, Oriel, etc. No
special machining is required: this design takes advantage of the
epi-illumination port available on most research microscopes equipped
for fluorescence. Best of all, it can be built by anyone familiar
with a microscope but with little prior experience in optics, and
this article should teach you most of what you�ll need to know but
by all means, feel free to learn more!
LASER BEAM-STEERING
Figure 1
illustrates how beam-steering, or scanning, is accomplished in a light
microscope. The same principles are involved regardless of whether
one is scanning the beam for a confocal laser system or moving an
optical trap. When properly set up and aligned, microscopes have
two different sets of planes, called here the image and
aperture planes. Planes within each set are said to be
optically conjugate to one another: when anything is in focus
in one of these planes, it is in focus in all the others. Thus, the
retina of the person viewing the microscope (or, for that matter, the
camera focal plane), the intermediate image, the specimen plane, and
the field diaphragm form one conjugate set: the image planes.
Likewise, the eyepoint of the eyepiece, the objective rear pupil, the
condenser aperture, and the lamp filament (the last being included
when using K�hler illumination) form a second conjugate set: the
aperture planes.
These two sets bear a special relationship to one another:
translation of an object (or its image) along an axis in an
image plane produces rotation of the beam about an axis through
an aperture plane, and vice versa. Thus, to steer a focused spot of
light in the specimen plane, one rotates it about an axis running
through an aperture plane. One particularly convenient plane to use
is the eyepoint, where rays of light focused in the image plane are
parallel. This scheme is used by several commercial scanning confocal
microscopes to scan the laser spot. An analogous approach will be
used to scan our optical trap.
BUILDING THE TRAP
Figure 2
shows a highly schematized layout for the trap. This design can be
adapted to work with most research microscopes equipped for
epifluorescence (Nikon, Olympus, Zeiss). Traps based on this design
have already been built successfully by the author on the following
microscope models: Nikon Diaphot (original model), Nikon Diaphot
200 & 300, Zeiss Axioskop, Zeiss Axiovert, and Zeiss Universal.
Microscopes that have a parallel light path behind the objective lens
are particularly simple to work with: these include most inverted
microscopes (e.g., Nikon inverted scopes, which have a negative lens
in the objective turret to produce parallel light), or those equipped
with objectives having a rear focal plane is at infinity (e.g.,
microscopes using Zeiss ICS objectives). With little or no
modification, this trap can be combined successfully with most
imaging modes, including brightfield, darkfield, phase contrast, and
differential interference contrast (Nomarski DIC). Phase
contrast is not particularly recommended for high power use, however,
since the phase rings inside the objective absorb a portion of the
laser light and cause heating. Passing a laser beam through the
Wollaston prisms used in DIC imaging poses no particular problems, by
the way.
Since this trap occupies the epi-fluorescence port, use of the
fluorescence mode is problematic. With some extra work, however, the
design can be altered to accommodate fluorescence illumination as
well. One way is to combine the infrared laser and short-wavelength
epi-illumination lights together outside the microscope and use a
special dichroic mirror in the filter cube (that reflects, say, both
blue and infrared but passes green and red in between). Another is
to alter the microscope to accommodate two, stacked dichroic filter
holders, one above the other, so that the laser can be introduced into
one and the epi-illuminator light into the other. In the latter case,
the conventional dichroic mirrors can be used.
The heart of the design is a simple 1:1 telescope arrangement that
is used to both steer and parfocalize the laser spot. Two identical
planoconvex lenses, L1 and L2, are placed the sum of their focal
lengths apart, so that parallel light in produces parallel light out
of the same beam size. (For alternative designs, see the review by
Svoboda & Block, 1994 in the References). By placing these lenses
with their flat surfaces facing one another, spherical aberration is
minimized without resorting to expensive aplanatic lenses. The
rearmost lens is mounted on an x-y-z translation stage or
micromanipulator. To a good approximation, movements of this lens in
all three dimensions generate corresponding movements of the laser
trap in the same three dimensions. Here�s how it works.
Movement in the axial direction occurs because, as L1 is pushed
towards L2 (defined here as the axial, or z-direction), the
parallel beam entering the telescope (at the back of L1) becomes
somewhat divergent after leaving L2. This pushes the focal spot
away from the objective and deeper into the specimen. Likewise, as
L1 is pulled away from L2 in the axial direction, the light from the
telescope becomes somewhat convergent, bringing the focus towards the
objective. Movement of lens L1 in the x-y plane, perpendicular
to the optical axis, produces a deflection in the light leaving the
lensrotating the beam, in essence. If lens L2 is imaged into
the back of the objective pupil, then this rotation occurs in a
conjugate plane to the objective pupil, resulting in a translation of
the laser spot, as described above. Lens L1 accomplishes this imaging
by virtue of its location at a distance 2f behind the objective
pupil, where f is the focal length of L1 (or L2). N.B: in
practice, this distance need not be strictly equal to 2f, and
one can get away with somewhat larger distance, although in that case
a portion of the laser light will be blocked by the back aperture of
the objective when the beam is steered away-from-center, diminishing
trapping strength.
In some designs, it may be desirable to produce additional beam
expansion in the telescope formed by L1 and L2, instead of doing it
all in the laser beam expander placed earlier in the optical train.
In this case, the focal length of L1 should be chosen to be less than
L2 (say, f /2 for a 2� expansion) and the position of L2 should
be adjusted accordingly.
List of Parts
(1) A microscope (either upright or inverted) equipped for
epi-fluorescence and carrying a high NA objective (Magnification =
40×100×, NA = 1.251.40, oil- or water-immersion
recommended). The microscope should have a holder or turret for a
fluorescence filter cube and a port for epi-illumination, but need not
have most of the other items associated with fluorescence work (e.g.,
filter sets, mercury arc lamp and power supply, additional
epi-illuminator optics, etc.). It should also have a TV camera port
(C-mount adapter) so that microscope images can be viewed on video.
(2) A dichroic mirror that reflects the appropriate laser
wavelength (and perhaps beyond, if desired), usually ~1100 nm or
~850 nm, but transmits visible light below 650 nm. This is sometimes
called a hot mirror. The hot mirror should fit into the
epi-fluorescence cube of the microscope. If such a mirror is not
available from the microscope manufacturer (often the case), it can
be custom-made by a company that specializes in manufacturing such
items, e.g. Chroma, Inc. (Burlington, VT) or Omega Optical (Burlington,
VT). Typical specifications of a mirror for a Nd:YAG laser are: 1 mm
thick, BK-7 glass, front surface R >= 99.5% @ 1064 nm, polarization
horizontal, T >=85% @ 546 nm, anti-reflection (A/R) coated on the
rear surface for 1064 nm, sized for (and optionally mounted in) the
particular microscope�s filter cube.
(3) A set of IR-blocking filters. For work at 1064 nm, use
KG-series optical glass made by Schott Optical (available in sets or
individually from Melles Griot, Newport, Oriel, Schott, and others).
KG-1, KG-2, KG-3, and KG-4 glass in 2 or 3 mm thickness work well as
variable attenuators of the infrared, without blocking much visible
light. For work at 850 nm, various colored Schott glasses can be
found that will do the job, or custom filters can be made, just as
for the previous item.
(4) Two plano-convex lenses L1 and L2 (or, if you will be using
a lens already inside the microscope as the fixed lens, one such
lens). These can be optionally anti-reflection coated for the laser
wavelength (more costly, but conserves laser light). Choose their
focal lengths according to
Figure 2 and as discussed in the text, but try to keep the focal
lengths between 30 mm (min) and 180 mm (max), with 50 mm being
typical. Most lenses come in 1 or 0.5 standard diameter:
either will do. A more costly alternative is to use aplanatic doublet
lenses instead of singlet lenses: these minimize spherical
aberrations. Purchase a mount for the fixed lens L2 that will hold
it in the appropriate position on the optical axis of the filter cube.
(5) A laser beam steerer, consisting of two 45° kinematic
mirror mounts oriented orthogonally over one another on a single
vertical post. Also purchase two mirrors M1 and M2 (metallic or
dichroic, your choice: dichroic mirrors are more costly but reflect
better) to place in these mounts. Laser beam steerers are available
as put-together units from most optical manufacturers. Buy a
relatively low-cost version that still offers fine mirror control.
(6) A three-axis translation stage attached to a holder for lens
L1. The stage should have at least 0.5 travel in x- and
y-directions and 1.0 in the z-direction. Most
micrometer-driven stages work quite well. If your budget is limited,
the z-travel can optionally be replaced altogether by simply mounting
the x-y stage on an optical rail, then sliding this stage
forward and back along the rail for z-control, since typical axial
movements are larger and the trap is less sensitive in this dimension.
The stage should be set up in such a way that lens L1 is on the
optical axis of the filter cube and positioned at a distance of
2f (where f is the focal length of L1 or L2) behind
L2.
(7) A variable attenuator. Attenuators come in various forms.
If the laser isn�t too bright, a simple rotating neutral density
wedge can be used, or a fancier dual-wedge compensating attenuator
(available from Newport and others): check the manufacturer�s
ratings for maximum power handling capability before purchase. If
the laser light is brighter and happens to have a fixed polarization,
a better attenuator can be made from a rotatable halfwave plate
followed by a fixed prism polarizer (a Glan-Thompson prism, Glan-Laser
prism, or polarization beam-splitting cube will do): simply turning
the halfwave plate changes the light level. A put-together version
of such an attenuator is also available as a commercial unit, but is
more costly. If the laser is a diode, satisfactory control of the
light level can often be achieved electronically by changing the
diode supply current above the laser threshold. Cautionary note:
certain diode lasers only produce a clean beam for a
narrow range of input currents.
(8) A laser beam expander. Beam expanders can be homemade from
singlet or doublet lenses, but the best solution is to purchase one
ready-made from an optical manufacturer. These are carefully designed
to minimize wavefront distortion at the desired wavelength over the
full aperture of the device, and therefore minimize spherical
aberrations, eventually leading to better focusing in the specimen
plane and thereby to better trapping. Beam expanders typically come
in fixed sizes (3×, 5×, 7×, 10×). Choose
a magnification factor that will approximately fill (or slightly
over-fill) the objective pupil of your microscope objective (assuming
you used a 1:1 telescope for L1 and L2).
(9) A momentary shutter. For safety, it�s a good idea to have
the laser beam off at all times when not trapping. A particularly
convenient solution is to place a normally-closed electronic shutter
in the beam near the laser, operated by a foot pedal. When the foot
switch is depressed, the trap is turned on. When it is released, the
trap is turned off. This leaves ones hands free for microscope
operation and beam steering. A Uniblitz shutter and associated
controller unit works well (Vincent Associates), and a foot switch
and cord can be obtained from any electronic supply house (Newark,
Sager, Radio Shack, etc.). These can be hooked directly together
without additional electronics. Buy a shutter with metallic
reflecting surfaces (and not Teflon or black absorbing surfaces),
so that the laser power doesn�t damage it.
(10) A laser suitable for optical trapping (see above). If
biological work is intended, a near-infrared laser wavelength is
recommended. If a diode laser is chosen, be sure that it operates
in single mode and that the beam has been properly circularized
using cylindrical lenses or anamorphic prisms. If a conventional
laser is chosen, make sure it operates in continuous-wave (CW)
fashion in TEM00 mode. If the laser beam quality is
particularly dirty, it may be necessary to spatially
filter the light, but this is usually unnecessary.
(11) A CCD videocamera for the TV port. This camera will be used
to view the laser light and align the trap. CCD cameras use silicon
as the photosensitive element, and therefore can see laser
light out to 1064 nm, beyond the usable range of conventional Vidicon,
Saticon, and Newvicon camera tubes. Many CCD cameras (Dage,
Hamamatsu, Pulnix, etc.) have a heat filter screwed into the camera
housing right in front of the CCD array: this should be removed
prior to use. An excellent alternative to the CCD is the Ultracon
(or silicon diode) tube, which also uses a silicon sensor, and is
especially suited for lower light level work (e.g., video-enhanced
DIC), or an extended-red Newvicon tube, but the latter is only barely
able to see 1064 nm. Yet another possibility is to use a budget B/W
CCD camera of the type used in surveillance for trap alignment and a
second, high-quality camera for imaging. Inexpensive CCD cameras are
available for under $200 that do the job well. Most of these
considerations don�t apply if a diode laser at ~850 nm is used, since
practically all TV cameras of interest are reasonably sensitive at
that wavelength.
(12) Miscellaneous mechanical pieces, including an optical rail
and associated posts, mounts, and clamps that can be used to hold
various items, such as the attenuator, beam expander, and shutter.
Also, be sure to purchase one or more IR sensor cards (available
from most optical manufacturers) that can be used to visualize the
position of the invisible infrared beam. The translucent variety
is the most practical, since it permits viewing the beam on both
sides of the card. For safety, obtain laser goggles that block the
laser wavelength (or make your own from IR-blocking filters, but
remember to cover the sides). You may wish to purchase a laser
power meter to measure the light levels at various stages during
alignment and experiments.
Putting it all together
Place the microscope on a solid, level, flat surface. An optical
table is best but certainly not required. Insert the high NA
objective in one position of the turret, as well as a low power
brightfield objective (say, 10×) in another, to be used for
rough beam alignment. Mount the laser at tabletop level as far away
as convenient from the microscope (give yourself room to work!) and
point it perpendicular to front-to-back axis of the microscope. Make
absolutely sure there is adequate room behind and to the side of the
microscope. Insert the IR-reflecting mirror inside the filter cube
and place it in the microscope. Insert an IR blocking filter under
the eyepieces (say, inside the binocular unit) for safety. If the
epi-illumination optics comes equipped with a built-in lens that
can serve the role of L2 (e.g., the lens at the end of the
epi-illuminator arm on the Zeiss Axioskop or the original model of
the Nikon Diaphot), you may consider using this same lens in its
usual position instead of providing your own. Otherwise,
remove the epi-illuminator arm (or at least parts of it), so
that a free optical path leads from the back of the objective to the
filter cube and on out of the microscope. Make sure there are no heat
filters in the way: these block infrared light (the old Nikon Diaphot
had one in the turret housing)! Similarly, make certain that there
are no polarizing filters in this path (some microscope carry
polarizers for DIC imaging between the filter cube and the objective,
but most are placed safely behind the cube). Crudely position lens
L2 a distance 2f behind the objective pupil, or as close as
possible to that position if it cannot be placed there (this is often
right against the rear of the microscope, or possibly just inside it)
at the correct height for the optical axis of the filter cube.
Mount and crudely position lens L1 and its x-y-z stage.
Mount the laser beam steerer and rough-align it: mirror M2 goes at
the same height as the optical axis of the filter cube, and mirror
M1 goes at the same height as the optical axis of the laser on the
tabletop. Follow the diagram in
Figure 2.
Position the attenuator, beam expander, and shutter on a single
line between mirror M1 and the laser. These three may be mounted
together on an optical rail, if desired. The exact order of the beam
expander, attenuator, and shutter is to some extent arbitrary.
However, for use at high power, and especially with neutral density
attenuators, it may be desirable to spread the laser light over a
larger aperture by placing the beam expander first, so as to prevent
optical damage. (The shutter, too, may require positioning after the
beam expander.) However, if a polarizing prism/waveplate combination
is used in this position, a larger hence more expensive
prism might be required. (Similarly, larger shutter apertures are
more costly and open/close more slowly.) Power levels permitting, it
may be advantageous to place both attenuator and shutter in front of
the beam expander, as shown in
Figure 2.
Alignment Procedure
Let's begin alignment. As you probably know, there are many ways
to skin a cat when it comes to aligning any optical system. The
method described here is tried-and-true, though, and contains several
useful tricks. IR lasers can be more difficult to work with because
their beams are invisible. Still, with a bit of care, exact alignment can be
accomplished fairly painlessly. In fact, several of the steps (chiefly those that verify
what's been done) can be skipped altogether once you get the hang of the whole process,
and alignment can be accomplished more rapidly. Plus, once the basic alignment is done,
most of the parts won't need to be tweaked further.
Turn on the microscope, and place a not-too clean glass microscope slide (with no
specimen or cover glass, but perhaps a bit of dust or a fingerprint or two on it) on the
stage. Focus the 10× low power objective on the slide's surface and align and center the
condenser as well, then turn off (or block) the microscope illuminator. Put on your laser
goggles! Always align the laser at the lowest possible power, so adjust the attenuator
accordingly. Now turn on the laser. We begin by aligning the first portion of the path,
which is the easy part. Beginning at the laser output window and using the IR phosphor card to see the beam, align the shutter, beam expander, and
attenuator so that the beam passes dead on through the
center of these devices (the beam expander is particularly important:
make sure the laser is on center both coming in and going out). Then
adjust mirror M1 of the laser beam steerer to send the beam
vertically.
Now we'll coarse-align the upper path. Temporarily i>remove
lenses L1 and L2, and turn the microscope turret to a position where
there is no objective screwed in. Verify that the filter cube is in
position. Lay the IR card on the microscope stage in the specimen
position, in place of the glass slide, and lower (or raise, if using
an inverted microscope) the condenser temporarily out of the way, but
don't touch the microscope's focusing knob. Now, adjust mirror M2 of
the beam steerer (and possibly M1 as well) until the light passes
into the microscope and reflects straight up through the specimen
plane vertically. You should also try placing the card lower, right
over the empty objective turret, and verify that the beam passes
through the center of the turret hole. Then insert lens L2 (only)
and position it in such a way that the light continues to pass
vertically up through the specimen and through the center of the
turret hole. The laser light should come to a focus somewhere
behind the turret hole at this stage. Swing back the 10×
objective. Once again, place the IR card in the specimen position
and adjust M1 and M2 as necessary to center the light. Insert lens
L1 and using the IR card, crudely adjust its x-y position by
eye in such a way that the light beam hits the lens at its exact
center. Fix the z-position of L1 using a ruler, placing it
2f behind L2, where f is the focal length of L1 and L2.
Swing the objective turret once more to an open position and place
the IR card back in the specimen position. This time, leaving M1
and M2 as they were, adjust the x-y position of L1 until a
uniform vertical beam emerges once again (checking again the centering
at the turret opening as well as the specimen).
We are now ready for fine-alignment of the upper path. Swing in
the 10× objective and return the condenser to its original
position. Place the IR card somewhere convenient behind the
condenser. If everything was done right, the card should be
illuminated with a nice, uniform disk of light once more.
If not, then adjust the x-y position of L1 until it is
(you may have to iterate on the steps above, but probably not if
you've been careful up to this point). You are now ready to
hunt for the beam on the TV camera. Shutter the laser
and turn on the microscope light, then verify that it is still in
alignment (both the objective and condenser) and focused on the
slide. Get an image on the TV monitor. Then turn off the
microscope light and un-shutter the laser. Don't look into the
microscope while the laser is on: use the TV image. (If you
previously placed an IR-blocking filter in the normal microscope
path, as shown in
Figure 2, remove it for now: this can be re-inserted when the
trap is all aligned). You should see a bright spot on the screen,
corresponding to where the laser light bounces off the slide (if not,
go back and re-do the above). You will often see various stripes,
spots, and other patterns at this stage, due to small amounts of
scattered light in the preparation, Newton's fringes in the glass,
and other interference phenomena. This is normal. The main laser
spot, however, is the brightest of all, and it disappears
when you're out of focusthe other patterns don't, by and large.
Center the laser spot in the field of view using the
x-y controls for L1. Now, using the z-control,
minimize the diameter of the spot: this should bring the laser
roughly into focus at the specimen plane. You may not find the
laser spot if the microscope has drifted a bit out of focus, so be
prepared to touch up the focus a bit during this procedure.
At this stage, we're ready for crude trapping and final
alignment. Shutter the laser again. Prepare a microscope specimen
that can be easily trapped: a suspension of either yeast or bacterial
cells at moderate density works best, or you can also substitute a
suspension of silica microspheres (0.2 to 1.2 mm diameter, whatever's
handy) or even blood cells. Latex microspheres can be used, but
these are not recommended, since they don't trap as easily in the
axial direction. Use a sealed grease ring or tunnel slide or flow
chamber or any other mount that allows the preparation to have some
depth, say 50100 µm or more, between the coverglass and
the slide. Put the specimen on the stage, then place a drop or two
of immersion fluid on it (oil or water, depending on your objective)
and swing in the high power objective. (If you are using an
oil-immersion condenser, place immersion oil on this as well).
Turn on the microscope, then focus and align it in the usual way
on the specimen. Adjust the position of focus to the top of the
chamber (to the bottom if an inverted microscope), on the interior
chamber surface nearest the objective lens (at the glass-water
interface). Turn off the microscope once more and un-shutter the
laser. Using the video camera, locate the laser spot, and center it
once more using the x-y controls of L1. If this is not
possible, it is probably because (1) the microscope is not focused
properly at the glass-water interface, or (2) the earlier alignment
procedure wasn't performed correctly.
Assuming that you can view the laser spot on camera, you are now
ready for the final alignment step, namely, walking the
beam until it is precisely coaxial with the optical axis
of the objective. This is done in a series of small, iterated beam
rotations (by means of lens L1) and translations (by means of mirror
M2) that ultimately a produce perfect, diffraction-limited spot.
First, turn the focusing knob gently up and down through the plane
of the glass-water interface. You will see the laser spot shrink
to a small circle at the interface, but expand to either side of it.
In general, this expansion will be asymmetric: for example, the
light will appear to come in from the right and leave to the left,
or in from the bottom and out towards the top, etc. The purpose of
this final adjustment is to cause the light to form a uniformly
circular set of rings that will collapse to a single point at
focus and expand back into a uniform circular set of rings beyond
it. This is accomplished by (1) moving the spot away from the center
of the field a bit, using L1 (or M2), but then (2) restoring it to
the center using either M2 (or L1). You'll have to determine which
to start with, the mirror or the lens: one choice will make things
worse, the other will make things better. The main point is to use
the opposite control for restoring the spot to the center to
the one just used for moving it away, and to keep doing this until
the light is well-symmetrized, as confirmed by focusing up and down
through the interface. This set of adjustments must be done twice:
once for the x-direction and once for the y-direction.
Be sure to use the corresponding pairs of knobs on L1 and M2 that
produce just the desired x- (or y-) movement. When
the beam looks circular and coaxial, you're done and ready to trap!
Turn the microscope illuminator back on and bring the laser up
to a higher power using the attenuator: you should begin to see
some of the objects in your specimen already caught by the trap
(unless their density is too low, in which case you should move
the microscope stage until something comes into view that can be
captured). Try moving lens L1 and the trapped object(s) ought to
move correspondingly. In fact, you should now be able to manipulate
the object anywhere in the field of view using the controls on L1.
Focus a bit below (above, if an inverted microscope) the coverslip
and try to trap a single object. Is it still in sharp focus? If
not, adjust the z-control of L1 slightly until the trapping
zone is perfectly parfocal with the specimen plane. This control
may require some tweaking for different kinds of objects, say,
silica beads vs. mitochondria, since these will trap at different
heights with respect to the laser focus. Congratulations: your
trap is fully aligned and operational!
As a last touch, you may wish to balance the relative levels of
IR and visible light reaching the video camera, so that the laser
trapping spot can be easily spotted against the background image,
but does not overwhelm the camera. One convenient way to do this
is to select an appropriate IR filter that blocks some, but not
all, of the laser light (see
Figure 2), and place this in the optical path of the microscope
somewhere between the back of the filter cube and the camera (inside
the C-mount adapter is one place, a substage slider in an inverted
microscope is another). Some trial and error may required to select
the proper balance.
Finally, recall that high power IR lasers are invisible and
potentially dangerous, especially if they are allowed to impinge
at full power upon your retina. Take safety precautions, including
placing IR blocking filters in parts of the optical path where IR is
not needed, e.g., before the eyepieces. Wear safety goggles while
aligning the trap. Post signs. Consider enclosing the external
beam path so that nothing can accidentally be placed into it. Above
all, use caution coupled with common sense.
VARIATIONS ON A THEME
There are many ways to soup up this design. If you
want to get fancier, you might consider one or more of these
improvements.
1) Use a motorized manipulator for lens L1 and control it using
knobs, a joystick, or a computer. While you're at it, you could
build a fancy computer-driven video overlay device that tracks and
displays the position of the trap, superposing it on the image on
the video screen at all times, and indicates if the trap is on or
off.
2) Use a piezoelectric stage positioner driven by a
computer-controlled voltage. This is especially helpful in
calibrating the forces produced by the optical trap against
Stoke's drag on particles, as well as with micromanipulation
tasks.
3) Consider motorizing the focus of the microscope, or, as an
alternative, moving the objective in the axial direction with a
piezoelectric device. You may also wish to automate the attenuator
and/or shutter.
4) Combine an optical trap with fluorescence imaging, using the
approaches discussed earlier, namely, superpose the trapping and
epi-illuminating lights, or use two stacked dichroic filter cubes.
5) Consider using the telescope L1/L2 only to parfocalize the
trap and the specimen, and use another potentially faster method
to steer the trap, for example: galvanometer mirrors, or
acousto-optic or electro-optic beam deflectors (see review by
Svoboda & Block, 1994).
6) Build a position-sensitive detector that can determine the
displacement of a particle with respect to the center of the optical
trap to nanometer (or better) recision. This detector can be based
on a quadrant photodiode or a dual-beam interferometer. The detector
can then be used to calibrate force accurately and thereby turn the
optical manipulator into a practical picotensiometer.
With a position-sensitive detector, it is further possible to produce
a closed-loop feedback device that can function as an isometric
tension clamp (see both Finer, et al., 1994 and
Svoboda & Block, 1994).
LEGAL DISCLAIMER
Lasers can cause damage to material and injury to people. These
instructions are provided for educational purposes only. While
effort has been made to insure safety and reliability, the author
assumes no liability whatsoever for damages consequent to any
activity related to these protocols.
ACKNOWLEDGMENTS
The author thanks Howard Berg, Christoph Schmidt, Karel Svoboda,
and Koen Visscher for helpful discussions and comments.
SELECTED REFERENCES
Block, S.M. Optical tweezers: a new tool for biophysics.
In J.K. Foskett & S. Grinstein, eds. "Noninvasive Techniques in
Cell Biology," New York: Wiley-Liss. Mod. Rev. Cell Biol.
9: 375-402 (1990). Slightly out-of-date, but a good all-around
introduction at the Scientific American level.
Block, S.M. Making light work with optical tweezers.
Nature 360:493-495 (1992). A short, accessible review of
basic principles and recent applications.
Finer, J.T., Simmons, R.M. and Spudich, J.A. Single myosin
molecule mechanics: piconewton forces and nanometre steps.
Nature 368: 113-119 (1994). A tour de force of optical
tensiometry.
Kuo, S.C. and M.P. Sheetz. Optical tweezers in cell biology.
Trends Cell Biol. 2: 116-118. (1992). Another short,
accessible review of basic principles and recent applications.
Simmons, R.M. and J.T. Finer. Glasperlenspiel II: Optical
Tweezers. Curr. Biol. 3: 309-311 (1993). A nice
write-up on how to do optical tensiometry using force feedback.
Svoboda, K. and Block, S.M. Biological applications of optical
forces. Ann. Rev. Biophys. Biomol. Struct. 23: 247-285 (1994).
This is the basic manual. If you want to get serious about
optical trapping, read this.
Svoboda, K. and Block, S.M. Force and velocity measured for
single kinesin molecules. Cell 77: 773-784 (1994).
This and the following paper demonstrate the power of optical
trapping combined with interferometry.
Svoboda, K., Schmidt, C.F., Schnapp, B.J., and S.M. Block.
Direct observation of kinesin stepping by optical trapping
interferometry. Nature 365:721-727 (1993). Companion to
the previous paper.
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