Download Optical tweezers using a diode laser

Optical tweezers
using a diode laser
Robert S. Afzal and E. Brian Treaty
Candela
Laser Corporation,
Wayland,
Massachusetts
01778
(Received 22 October 1991; accepted for publication
16 December 1991)
Simple modifications were made to a commercial microscope to enable injection of light from
a diode laser, and demonstrate optical tweezers action. The basic properties of microscope
I optics are presented together with discussion of principles to be followed in arranging the
external optics for achieving useful tweezers. Procedures using a single-mode diode laser
along with experimental results are presented in enough detail to permit readers to make their
own system for trapping and manipulating single cells. It is surprisingly easy to
demonstrate tweezers action once some basic concepts are understood.
I. INTRODUCTION
Optical tweezers’ are a device used to manipulate
small objects such as biological cells using a focused laser
beam. The single beam gradient force trap was originally
demonstrated by Ashkin and co-workers2 In its most common form, optical tweezers apparatus consists of a laser
beam introduced into a microscope and focused by the
objective into the object plane. Objects are trapped near the
focus of the beam and can be manipulated by moving the
laser beam. Once a laboratory curiosity, optical tweezers
are gaining acceptance as a useful laboratory instrument.
Optical tweezers have been used in various fields of
biophysics and biotechnology. Aside from simple uses such
as holding one cell or organelle and moving it relative to
the other cells, tweezers have been used to make precise
force measurements on the compliance of bacterial
flagellae3 and other biological motors.4 Tweezers have also
been incorporated
in cellular surgery and fusion
experiments,5 as well as in automated cell counters and
sorting apparatus.6 Further applications await the invention and development of suitable techniques by a cadre of
researchers with optical tweezers available to them.
To date, most tweezers incorporate Nd:YAG, or an
argon-ion laser for the trapping beam, which tend to be
large and expensive laboratory devices. The use of diode
lasers for trapping7 not only provides a cost cutting advantage, but the mechanical support can be smaller and
lighter, making it easier to move and set up the instrument.
This will hopefully make this apparatus more accessible to
a larger number of potential users.
In this article we describe how to construct a simple
tweezer device based on commercially available parts and
provide the reader with a working knowledge so that modifications and customization of this basic design can be
made. In the second section we describe the microscope,
provide a simplified theory and discuss the requirements
for beam steering in order to understand how to couple the
laser beam effectively into the microscope. The third section discusses the trapping requirements of the laser beam
used. Section IV describes the diode laser used and how to
power the diode. Section V describes how to couple the
beam into the microscope and presents some results from
our own experiments.
2157
II. THE MICROSCOPE
A. Description
The microscope used in this work is the Nikon Diaphot TMD. This is an inverted microscope with the objective lens below the stage, but the regular type (overhead)
can also be used. It has three auxiliary ports-the
video
output port, the epi-fluorescence attachment port, and the
35 mm camera port. The video accessory is very important,
in that we made more adjustments and measurements
while looking at the video display than while looking
through the eyepieces. Laser beams were injected through
the epi-fluorescence port. The video camera, a Sony XC57, has some sensitivity in the IR so the location and symmetry of the laser beam (where it intersects the glass-water
interface below the object plane) are readily checked.
The microscope objective used was a 100X oil immersion (CFN Plan Achromat, N.A. 1.25, working distance
0.16 mm, focal length 1.71 mm). (Better visibility is available by using phase contrast.)
The regular eyepieces ( 10 X CFWN) were modified by
attaching infrared blocking BG 18 glass filters to their
lower ends for eye protection, supplied by Grayco Optics.
The epi-fluorescence attachment (without the exciting
lamp) comprises a focusing lens (f-160
mm) and an
adjustable iris in the field diaphragm plane. The epi-fluorescence dichroic filter assembly was modified by replacing
the filter glass with a visible-IR dichroic supplied by CVI,
and the laser beam was injected through the epi-fluorescence port, sometimes with the epi-fluorescence attachment plus an external eyepiece, and sometimes without.
The dichroic was the only specially coated optical element*
in the microscope.
We used a video adapter with relay lens in conjunction
with a Sony XC-57 camera for video monitoring and a
Tektronix oscilloscope camera to record pictures of the TV
monitor, and a Nikon 35 mm camera to take color or black
and white photographs.
The various ports referred to are depicted in Fig. 1.
B. Simple theory of microscope
The microscope manufacturers do not supply optical
diagrams or other details relevant to the design of their
instruments. Fortunately, one can work out all the details
Rev. Sci. Instrum. 63 (4), April 1992
0034-6746/92/042157-07$02.00
@copyright,
1992 American
Institute of Physics
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see http://rsi.aip.org/rsi/copyright.jsp
Inverted Microscope
FIG. 2. Simplified optics of objective and eyepiece.The spots at A and B
are minimum in size when the back aperture is filled. The eyepoint is
located at the image of the objective back aperture formed by the eyepiece. Light beams passing through A ’ and B’ are in different directions if
the back aperture is filled in both cases.The planes (A, B) and (A’ and
B’) are conjugate, as are the eyepoint and the objective back aperture.
Foc”sin~
K m %I(
,
smge
-
ini
sample
-we
I
I
LA
/
EY&Yan&d
"lew
Of
m*
stqp
Back
Of
m*
Ob~tlve
and
*,ccllYe
oQp?cl~ve/--
FIG. 1. Schematic of typical inverted microscope showing where laser
beam is injected.
necessary for injecting a laser beam for trapping by noting
the following simple facts and approximations:
( 1) The microscope objective is designed to form its
image at a distance of 160 m m from its back aperture plane
which is approximately at the position of its back focal
plane. A beam injected into this back aperture with a divergent radius of curvature of 160 m m (i.e., approximately
collimated) will focus to a point in the object plane. The
illuminated spot at this point will have minimum size if the
injected beam fills the back aperture. To move a spot of
minimum size across the object plane requires that the
approximately collimated beam at the back aperture be
steered in angle while always filling the aperture. Moving a
point source (from a focused laser beam for example) in
the objective image plane (at the 160 m m position) will
cause motion of the spot in the object plane, but its size and
intensity will vary unless the beam from the point source
always fills the back aperture. (See Fig. 2.)
(2) Various planes in the microscope are sequentially
imaged to one another and are said to be conjugate. There
are two important sets of conjugate planes: the field planes
containing the object (specimen) plane, and the aperture
planes containing the back aperture of the objective. (The
complex light amplitudes in these two sets are approximately Fourier transforms of each other.) A “collimated”
laser beam at any aperture plane appears at the back aperture of the objective and its position and steering there
are related to its position and steering at the other aperture
planes through simple imaging in conjunction with Abbe’s
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Rev. Sci. Instrum.,
Vol. 63, No. 4, April 1992
sine condition. lo (B earn diam.~sine of half-angle is preserved. )
(3) The optical train from the objective out through
the epi-fluorescence attachment can be seen from Fig. 1
and contains the following components. There is a negative
lens near the objective that moves the image at 160 m m to
infinity. A dichroic reflector at 45” couples the beam to the
epi-fluorescence attachment. It is thus sitting in a collimated beam. The first element encountered in the attachment is a positive lens that undoes the collimation and
allows the image to form in the field aperture plane of the
attachment. The lens focal length is about 160 mm. An
external eyepiece can be used outside the epi-fluorescence
attachment to examine the specimen in the object plane.
The conjugate to the objective back aperture (imaged by
the eyepiece, in conjunction with the positive-negative lens
pair enclosing the collimated portion of the beam) is called
the eyepoint. The eyepoint also identifies the position of the
Gaussian beam waist of a laser beam injected into the microscope that is focused on the object plane. (This is because the “waist” identifies the minimum in the beam diameter, which occurs at each aperture plane.) This
eyepoint is a good place to locate a steering mirror for
injecting a laser beam. (See Fig. 3.)
(4) For purposes of elementary calculation, ignore the
presence of the positive and negative lens pair forming the
collimator and simply assume that the image plane is 160
m m behind the objective back aperture. An example will
Eyepiece
Laser Seam
..
\
FIG. 3. Rotation of mirror located at eyepoint accomplishes the beam
control illustrated in Fig. 2.
Optical tweezers
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2158
make this clear. We want to inject a Gaussian beam with
spot size (l/e” intensity radius) 2.5 mm into the back
aperture of the objective” using a 10X eyepiece outside
the field stop of the epi-fluorescence attachment. The 10
Xeyepiece has a focal length of 25 cm/lo. Its distance
from the back aperture is then (16 + 2.5) cm. The eyepoint location is l/( l/2.5-1/18.5),
or 2.89 cm outside the
eyepiece. The image of the 6-mm-diam back aperture of the
objective has a diameter of 2.89/18.5 times 6 mm, or 0.94
mm. A Gaussian beam running backward from the objective starts with converging wavefront radius 160 mm and
spot size 2.5 mm at a wavelength of say, 842 nm. It propagates 18.5 cm, then encounters a 2.5 cm lens and forms a
waist computed to be 2.89 cm beyond the lens (eyepiece).
The computed waist size (radius) at the eyepoint is then
calculated to be 0.39 mm. The half confocal parameter of
this input beam (i.e., the distance from the waist at which
the Gaussian beam area is double that at the waist) is 55
cm, so there is no need to try to locate the waist exactly
and place it at the eyepoint; although the steering mirror
should be located accurately there. With the epi-fluorescence tube removed, a collimated beam with waist 2.5 mm
injected into the epi-fluorescence port does essentially the
same thing, but without the ease of steerability. This beam
encounters first the dichroic beam splitter, then the negative lens, and then the back of the objective.
C . Verification
of the beam steering
requirements
Looking into the microscope eyepiece one sees the circular field of the specimen plane. The. eye’s pupil is located
at the eyepoint. An injected beam is to be rotated about a
point in this eyepoint plane (outside the epi-fluorescence
attachment) so as to access any point in the circular field
of the specimen. It is easy to verify the steering requirements and measure the mirror scan needed.
Measurements were made with a green He-Ne laser.
Using a 10 cm focal length lens we focused the beam onto
the field diaphragm plane of the fluorescence attachment.
The lens was mounted on a micrometer driven stage so as
to move the focussed spot sideways in the field plane. The
object used in the object plane of the microscope was a 0.1
mm scale from a viewing loupe (Edmund Scientific). Two
different objectives were used in the microscope: IOX and
40 X . Motion in the object plane is reduced from that in
the field diaphragm plane by the objective magnification.
We measured this factor to be 11.05 for the 10 X objective
and 42.7 for the 40X objective. This means that the specified magnification are approximate. The field of view
through the regular oculars is 1.9 mm wide using the 10X
objective and 0.5 mm with the 40x.
The field number for these oculars is nominally 20,
which when divided by the objective magnification gives
the size of the field of view in millimeters. Using the 10X
eyepiece, a mirror was mounted on a rotating stage with
the axis passing through the eyepoint. As the mirror was
rotated, observations were made of the illumination at the
location of the objective back aperture.12 If the axis of the
rotating stage, arranged to be in the mirror plane, is not in
the eyepoint plane, rotation causes the green laser spot to
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Diameter
Of
Object Fteld
Negative
L?“S
Dtchrotc
Attachment
FIG. 4. Straightened-out version of the optical train between the object
plane and the beam steering mirror behind the eyepieces.A beam steering
of 46”going into the eyepieceproduces a 7.2”steering at the back aperture
of the objective, and this in turn moves the laser spot across the diameter
of the object field.
move off center at the objective back aperture; observation
through the oculars of a ground glass slide in the object
plane shows the point of green light fading as it moves t
away from the center of the field, eventually disappearing
before reaching the visual boundary if the offset is large
enough. With the mirror correctly situated at the eye point,
the intensity of the green spot is maintained all the way to
the visual boundary.
By rotating the mirror in the eyepoint plane about an
axis normal to the plane of incidence and viewing the HeNe spot in the object plane, one measures the required
angular motion of the mirror. For example, with the 10X
objective, and a 10x eyepiece behind the field diaphragm
plane, we measured 12.06”/mm for the mirror, or 24.12”/
mm for the beam at the eyepoint. Thus, to cover the 1.9
mm field requires 46“ of beam steering (23” of mirror rotation). Similarly, with the 40 X objective we found 49.4”/
mm for the mirror steering and 23.25” to cover the 0.5 mm
field. Thus with either objective, we require roughly 46” of
beam steering to cover the visual field, independently of the
magnification of the objective. The corresponding angle at
the back aperture of the objective is 7.2“. (See Fig. 4.)
(This independence is not surprising since the angular field
observed through the oculars does not depend on which
objective is in use.) The required mirror motion is half of
this for the rotation axis described above, and higher for
other rotation axes.
It is easier to inject a large collimated beam into the
fluorescence port than to connect the epi-fluorescence attachment, with an extra eyepiece and then introduce the
correct Gaussian beam waist at the eyepoint. The approach
is satisfactory if one chooses to move the microscope stage
for bringing particles into the beam. However, it is difficult
to move the stage as smoothly as one can move the laser
beam. If one wants maximum power delivered to the object
plane or wants to move the focal spot while maintaining
constant power, the epi-fluorescence attachment plus eyepiece are recommended. If less than maximum power is
satisfactory, lateral translation of the final lens of the collimator will provide some angular scan, and it is necessary
to overfill the back aperture of the objective to keep it
illuminated as the beam scans.
III. TRAPPING REQUIREMENTS
The trapping force2 is proportional to laser power for
constant beam parameters. This is basically because the
Rev. Sci. Instrum., Vol. 63, No. 4, April 1992
Optical tweezers
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forces are of the type pgrad E. Ashkin and co-workers2+”
have carefully distinguished amongst pressure (due to reflection and absorption), and the gradient forces (due to
refraction), and radiometer forces due to thermal gradients. (Optical trapping occurs where the net gradient and
radiation pressure balance each other.) The forces tend to
be perpendicular to the particle surface, which is readily
understood by considering particles large enough that the
optical field near the surface is separable into incident,
reflected and refracted componentsi
The continuity of
phase along the surface expressed as the conservation of
the tangential component of k vector leads to the laws of
reflection and refraction. Then the relation between k vector and momentum (multiplication by Plan&s constant in
the photon picture, for example) tells us that there is no
tangential momentum transfer; and so the forces are normal to the surface.
Laser light that has been injected through the back
aperture of the objective can be pictured in the object plane
as a set of rays filling a cone, but because of the finite spot
size at the apex, a set of Poynting vector flow lines would
be more appropriate. A particle that is small compared to
the spot, and located close to its center sees a roughly
parallel set of flow lines and will be propelled through the
focal point without trapping. A larger particle can experience the gradient forces and be trapped. Clearly the gradients are greater for larger N.A. With a N.A. of 1.25 the
cone (semivertical) angle is 70” in water while for N.A. of
0.85 it is 40”. We have observed that polystyrene spheres of
2 and 3 pm can be trapped with an objective having N.A.
0.85 (the 60xCF plan achromat with corrector) but 1 pm
particles cannot (i.e., at a power level of the order of 10
mW), whereas the 1 ,um particles can be trapped using the
N.A. 1.25 objective. With the latter objective, a minimum
section of the back aperture has to be filled before the 1 ,um
particles get trapped; otherwise, they are pulled into the
central part of the field and propelled out roughly vertically. The samples are usually enclosed between a slide and
cover with a ring of grease forming the containing walls. If
the focus is raised so as to approach the slide, there is a
possibility of trapping against the barrier formed by the
slide. This type of trapping has been observed.
Rays that pass close to the center of a spherical particle
produce momentum kicks due to refraction that are almost
equal and opposite as shown in Fig 5, which also shows
how rays further from the center produce momentum
changes with a resultant backward component. In a cone
of rays, those close to the axis therefore contribute little to
the trapping, but much to the radiation pressure (associated with reflection and absorption) and heating. (Particle
trapping involves a balancing of the refractive or gradient
forces and the radiation pressure.) Heating should also be
minimized in order to reduce any adverse effects of the
laser light on living cells. For this reason, Ashkin and
Dziedzic” recommended using a laser with a wavelength
in the range of 0.8-1.8 pm. Too long a wavelength will be
absorbed by water. Diode lasers are readily available in the
wavelength range 0.80-0.85 pm.
It is surprisingly easy to trap larger particles (-5-10
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Rev. Sci, Instrum.,
Vol. 63, No. 4, April 1992
Force
Incident Rays
Incident Rays
(b)
FIG. 5. Illustrates how rays near the edges of the beam can produce
backwards directed gradient forces while those near the center cannot.
The forces in each area are the resultants of the normal forces at the
points of refraction on each ray.
pm) such as yeast cells. Smaller particles (- 1 pm)
present more of a challenge and force one to check the
beam propagation at various stations in the optical train,
Trapping can be readily achieved by removing the fluorescence attachment and injecting a collimated beam directly
onto the dichroic beam splitter. Verification of trapping
can be made by moving the stage and observing all objects
move relative to the trapped object. Moving the stage is all
right for coarse motion but for fine control or for making
precise measurements, moving the laser beam is desirable
and more sophistication in the optical train is needed. The
approach we have followed is to add the fluorescence attachment and lOXocular, and place a movable mirror at
the eyepoint of the ocular as in Fig. 3.
Finally, since the particle is trapped beyond the laser
focus, and the trapped particle is to be observed in focus
visually, the laser beam has to be slightly convergent in the
region of the dichroic beam splitter in order for the trapped
particle to be brought into focus. (Note also that dispersion in the objective increases this requirement.)
IV. DIODE LASER DESCRIPTION
Diode lasers can be procured as single-mode or multimode devices. The multimode versions have higher power
and lower unit cost (per watt) and they are useful for
pumping solid state lasers, which may then be used for
optical tweezers. So far we have not found them useful
directly for optical tweezers. The single-mode laser, aithough having less power, is more suitable for tweezers.
Diode lasers have a larger beam divergence transverse to
the junction than along it. For example, the SDL-5412Hl
(Spectra Diode Labs 100 mW single mode) has FWHM
beam divergence angles of 30” and lo”. The emitting junction dimensions are 1 by 3 pm, and in each of the two
transverse directions propagation follows regular Gaussian
mode optics behavior.16 The equivalent waist positions are
different for the two directions, exhibiting astigmatism. By
Optical
tweezers
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2160
imaging the diode output with magnification using a high
N.A. collimating lens, one can fmd the vertical waist then
move the diode with a micrometer drive to bring the imaged horizontal waist to the same position. The difference
in micrometer readings is then a measure of the astigma-
tism. A typical value is 15 ,um for the single-mode diode
laser. Upon collimation (Melles-Griot No. 06-GLC 001
collimator) the two divergences are in proportion to the
junction dimensions, so an anamorphic prism pair (MellesGriot No. 06 GPA 004) is used to expand the beam (3 X )
F~~f-b’ws
L
..-5K
1
2N2222
5
Lamp
"1,
:~
i
+ 12”
47
OP400
FIG. 6. Circuit diagrams for (a)
powering the laser diode, (b) its
thermoelectric cooler, and (c) the
photddiode for measuring output
power. All capacitor units are PF
and the OP4OO’swere powered by
a + and - 12 V dc level.
PHOTODIODE
Inside Diode Laser
2161
2161
Optical tweezers
Rev. Sci. Instrum., Vol. 63, No. 4, April 1992
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and thereby reduce its divergence in the junction direction.
(The diode laser output is polarized parallel to the junction
and so the first surface encountered by the beam in the
expander is at Brewster’s angle.) Thus, one obtains a collimated, roughly circular, Gaussian beam (with some
astigmatism). This beam can be examined with a Reticon
array or a Spiricon laser beam analyzer.17 The wavelength
is typically 840 nm. The diode with its protective circuitry
can be mounted into a small package and mounted alongside the other optical elements, and fed through a short
cable from its power supply. Figure 6 shows a suitable
circuit diagram for powering the diode, Manufacturers’
recommended handling procedures must be followed to
avoid damage to the diode from electrostatic discharge.
V. EXPERlMENTAL
CONFIGURATION
Microscope
Objactiw
Epi-!aoreocencs
Attachment
II I
10x
Eye Piece
Colllmafor
Eye Point
\
0-m
\
4:l Telescope
Rotating
Mirror
$
/
\
\
Ansmorphlc
Prism Pair
FIG. 7. System optical layout for making force measurements.
2162
Rev. Sci. Instrum.,
4.0=10*’
3.5 *Ias
i60
3.0 r1 o-7
140
2.5 11 O-7
120
2.0 “I o-7
100
1.5.10“
80
I,0 *to-7
60
3.5
4
4.5
5
5.5
6
6.5
Incident Laser Power (mw)
FIG. 8. Measurement of trapping force on 2 p latex spheresas a function
of laser power at 842 nm wavelength, using Ashkin’s viscous drag
method.
AND RESULTS
Before getting involved in the development of a complicated optical train, it is very convenient and easy to
demonstrate trapping simply by removing the epi-fluorescence attachment and injecting the circularized laser beam
from the diode into the microscope onto the dichroic reflecting the beam through the objective. Confirmation of
trapping is easily done by slowly moving the translation
stage and observing all objects in the field of view move
relative to the trapped object. This allows the user to gain
a feeling for the optical trapping phenomenon and the
forces involved. In order to beam steer for the purpose of
moving the trapped object to a desired location or to make
more quantitative force measurements utilizing the viscous
forces, for example, a more involved optical train needs to
be developed. For these purposes, the epi-fluorescence attachment without the UV lamp was inserted into the back
end of the microscope. As stated earlier in this report, the
epi-fluorescence attachment and a 10X eyepiece projects
the image of the back aperture of the objective to the eye
point. Based on the Gaussian beam calculation, a beam of
about 0.8 mm diameter at the eyepoint will just fill the
back aperture of the microscope objective. To achieve this
we used a 4:1 telescope to reduce the beam diameter from
z 3 to -0.7 mm. We used two positive lenses, f = 10 cm
and f = 2.5 cm to form the telescope. Location of the laser
spot in the video field of view is determined by adjusting
the objective focus and observing the formation of a bright
i-l
I-J-
,
180
Vol. 63, No. 4, April 1992
Diode
Laser
spot on the video monitor. The spot is formed when light
reflected from the glass-water interface is imaged onto the
CCD array of the camera. Changing the separation between the lenses in the telescope allows the trapped particle
to be brought into and out of focus in the object plane.
Since objects at the eyepoint are imaged to the back aperture of the objective a gimbal mounted mirror placed at the
eye point can then steer the beam across the field of view
without steering off the back aperture of the objective. The
reader may then choose to beam steer by moving the gimbal mirror by hand, by motor driven gears or by galvanometers depending on the application desired.
To quantify our trapping capability we performed
trapping force measurements on 2 pm latex spheres. Measurement of the trapping force by the usual method13 of
finding the maximum speed that the particle can be held
against the viscous force (the 67rav of Stokes’ law) requires that the focus remain good and uniform as it is
moved over the field. In our experiment we used a rotating
mirror with a=14” wedge placed at the eyepoint (Fig. 7),
As the mirror rotated, the beam traced out an ellipse of 55
and 45 ,um major and minor axes in the object plane. We
could then determine the speed at which a particle was
released from the trap by measuring the angular velocity of
the motor. Maximizing the force with the available laser
power from the diode mostly involves minimizing the optical losses (especially by vignetting). Figure 8 shows a
graph of trapping force vs laser power. Power levels were
measured at the back of the objective by placing an object
with an opening the same size as the back aperture where
the objectives screw in. We used optics and equipment that
were available to us cheaply and quickly. Therefore. Gptical losses in the optical path could be greatly reduced by
using specially coated lenses and mirrors if higher trapping
forces are desired using the same laser.
We would encourage the interested reader to build and
experiment with optical tweezers, paying proper attention
to eye safety, and verify how easy it is to trap and move
particles. Once built, various improvements can be added
for beam scanning, computer driven manipulation, a beam
rotator (dove prism) for rotating ceils, and so on.
Optical
tweezers
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2162
ACKNOWLEDGMENTS
We thank Mr. Zhengliang Lu for setting up the diode
laser assembly; Dr. S. M. Block of the Rowland Institute of
Science for introducing us to the subject; Dr. Horace Furumoto for his interest, encouragement, and support; and
the National Science Foundation for funding this work,
under Small Business Innovation Research Grant No. ISI9060563.
’For a comprehensivereview, see S. M. Block, Noninvasive Techniques in
Cell Biology (Wiley, New York, 1990), Chap. 15, pp. 375-602. The
term “optical tweezers”was coined by A. Ashkin and J. M. Dziedzic in
their article in Science 235, 1517 ( 1987).
‘A. Ashkin, J. M. Dziedzic, J. E. Bjorkholm, and S. Chu, Opt. Lett. 11,
288 (1986).
?I. M. Block, D. F. Blair, and H. C. Berg, Nature 338, 514 (1989).
*S. M. Block, L. S. B. Goldstein, and B. 3. Schnapp, Nature 348, 348
(1990); A. Ashkin, K. Schtitze, J. M. Dziedzic, U. Euteneuer, and M.
Schliwa, Nature 348, 346 ( 1990).
‘R. W. Steubing, S. Cheung, W. H. Wright, Y. Numajiri, and M. W.
Bems, Cytometry 12, 505 (1991).
‘T. N. Buican, M. Smyth, H. A. Crissman, G. C. Salzman, C. C. Stewart, and J. C!. Martin, Appl. Opt. 26, 53 11 ( 1987).
‘S. Sato, M. Ohyumi, H. Shibata, H. Inaba, and Y. Ogawa, Opt. Lett. 16,
2163
282 (1991). The diode that we used had a wavelength of 0.84 pm.
‘The fluorescence attachment comes with a dichroic filter that reflects
shorter wavelengths from the exciter and transmits the visible ftuorescence. The optical element should be replaced by another dichroic that
reflects the infrared laser light and transmits the visible. Ours was fabricated by CVI. Some infrared still passesthrough the filter from the
sample, and this is useful for alignment using the video system, but eye
protection filters have to be added to the eyepieces.
‘See, for example, S. Inou6, video Microscopy (Plenum, New York,
1986).
IoF. A. Jenkins and H. E. White, Fundamentals of Optics (McGraw-Hill,
New York, 1951).
“The 2.5 mm radius is arbitrary. The back aperture of the objective is
about 6 mm. If the spot is too small there is no trapping, while too big
a spot over fills the aperture and causes circular fringes around the
central spot in the object plane, which represent wasted light.
“It is easy to remove the objective and place a sheet of translucent
material at the position of the back aperture.
13A. Ashkin, Phys. Rev. Lett. 24, 156 (1970).
14W. H. Wright, G. J. Sonek, Y. Tadir, and M. W. Berns, IEEE J.
Quantum Electrons 26, 2148 (1990).
“A. Ashkin and J. M. Dziedzic, U. S. Patent No. 4 893 886 (16 January
1990).
16H. Kogelnik, Bell Syst. Tech. J. 44, 455 (1965).
17Theseunits are available from EG&G and Spiricon, Inc., respectively.
Optical tweezers
Rev. Sci. Instrum., Vol. 63, No. 4, April 1992
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