Laser Trapping of

Micrometer-Sized Objects
Kathryn Child
Philip I. Thomas
Department of Physics
Washington University in St. Louis
6 December 2012

Abstract
We characterized the motion of micrometerscale beads and blood cells undergoing
Brownian motion and under trapping by an
optical
tweezer
apparatus.
Diffusion
coefficients and Boltzmanns constant were
calculated based upon variations in the
bead and cell movements.

Introduction and
Background
Brownian Motion
Brownian motion describes the random
movement
of
small
particles.
The
phenomenon is named for biologist Robert
Browni, who is credited with first describing
the phenomena while observing the random
motion of pollen in solution. In addition to
small particles in solution, models for
Brownian
motion
suitably
describe
numerous other processes, ranging from
molecular movement to stock market prices.
Numerous
mathematical
models
for
Brownian motion exist, mainly in the field of

particle theory. Classical mechanics fails to

adequately describe the high number of
interactions between multiple particles.
Thus, most modern models take a
thermodynamics-based, statistical approach
to quantification of multiparticle systems.
This is rooted in the Second Law of
Thermodynamics ii , which describes the
increasing entropy of a closed system.
When analyzing one particle, Brownian
motion is suitably modeled as a random
walk. Random walks originate in term as the
seemingly random mix of forward and
backward steps by a drunk. The term was
coined in 1905 by Karl Pearson to describe
random motion in discrete steps of a fixed
length iii .
These discrete steps form a
binomial
distribution
that
may
be
approximated as a continuous normal
distribution as the number of steps
increasesiv. Thus, when analyzing a single
particle undergoing Brownian motion, its
displacement may be modeled as a
Gaussian distribution.

Diffusion
Diffusion is a random process modeled as a
series of differential equations called Ficks
Equations.v

Child & Thomas 1

A diffusion coefficient for two-dimensional

motion may be calculated as a function of
the statistical average of the displacement
in each dimension, as shown in Equation 1.
D=<x2+y2>/(4t)

(Equation 1)

Boltzmann Constant
The Boltzmann Constant is named for
Ludwig Boltzmann, an Austrian physicist
who made important contributions to kinetic
theory. The number originates from gas
theory, where the constant was first
quantified as the gas constant divided by
Avogadros number. However, the constant
is fundamental in statistical mechanics for
relating entropy to the number of
microstates.
Thus, the Boltzmann Constant is important
when describing Brownian motion because
it
bridges
the
second
law
of
thermodynamics with physical motion.
The Einstein Relation of Kinetic Theory,
specifically the Stokes-Einstein Equation,
allows us to relate the statistical average
displacement, the Diffusion Coefficient, with
Boltzmanns Constant vi , as shown in
Equation 2.

Figure 1: Snell's Lawvii

(Equation 3)

In Equation 3, n1 and n2 are the indices of

refraction, 1 and 2 are the angles form
the normal of

the
incident
and
refracted
wave,
respectively as diagrammed in Figure 1.
(Equation 2)

Refractive Index
Upon encountering a sudden change in a
mediums properties, waves reflect. This
reflection may be composed of transverse
and longitudinal components. The behavior
of these reflections and components is
described by Snells law, which relates
angle of incidence and refraction for a wave
at a boundary, per Equation 3.

The behavior of waves at boundaries

derives from LaGrangian mechanics, which
calculates the probable path as a function of
kinetic and potential energy. For light
specifically, this related as Fermats
Principle.viii Hence, the motion described by
Snells Law is an optimization for minimum
energy transversal of a boundary. ix

Child & Thomas 2

Dielectrics
An insulator that may be polarized with
an electric field is a dielectric. x The
non-conducting object thus forms an
electric dipole as charges distribute
themselves in the object. When a
beam with an electrical gradient is
applied to a dielectric, the interaction
between the dipole and the dielectric
create a force.

Figure 2: Optical Trapping with Spring Model

Optical Tweezers
Optical tweezers use optics to create a
force on dielectric objects.xi A focused beam
of light may thus be used to hold a
microscopic object in place across three
dimensions. The 1997 Nobel Prize in
Physics was awarded to
Steven Chu,
Claude Cohen-Tannoudji, and William D.
Phillips for their work on trapping neutral
atoms.xii
Objects are able to be trapped due to the
presence of an electric gradient that is
strongest at the narrowest point of the beam.
Thus, a dielectric object with the
aforementioned presence of an electric
dipole experiences a restoring force to the
center of the trap due to the energy level.
This restoring force may be approximated
as Hookean in models. xiii Momentum is
conserved through Snells law. Specifically,
the change in momentum of photons
diffracted through the change in medium
between the surrounding medium and the
object provides the force that drives the
object to the center of the trap. At the center
of the trap, the angle of incidence is zero,
hence there is no diffraction, no change in
momentum of photons, and no net force.

Figure 2 xiv shows a basic optical trap,

including its relation to a Hookean spring
model. Optical tweezers have become an
important tool in varied areas of science due
to their ability to study the mechanics and
dielectric properties of microscopic objects
and even some sub-micron objects. For
instance, a team at MIT was able to attach
then subsequently optically trap micron
beads at the ends of a molecule of DNA,
then calculate the Hookean spring constant
of a molecule of DNA by manipulating it with
the optical traps.xv

Microscope
Microscopy is the application of one or more
refractive surfaces in order to magnify the
view by manipulating photon paths using
Snells law. xvi In a basic example, a curved
lens is used, and based on the curvature,
light takes different paths through the glass
due to the changing incident angles at
different points on the curved lens.
An important property of microscopes is the
focal length, which describes the distance
from the lens where light is focused. As

Child & Thomas 3

magnification increases, focal distance

decreases. Thus, at high magnification, the
use of an oil-immersion lens becomes
beneficial. The refractive index of oil,
relative to air, allows for a shorter focal
length.
The purpose of this experiment is to
understand optical tweezers and their use in
isolating particles, as well as to characterize
Brownian motion in two dimensions by
observing through a microscope the motion
of the beads and quantifying their
movement.

Methods / Procedure
Using an optical tweezers setup based upon
that of Bechhoefer and Wilson xvii , and
shown in Figure 3, the experiment used a

comparison of deterministic and random

motion to evaluate the diffusion coefficient
of both 1m and 3m beads and
Boltzmanns constant. 1m beads were
selected on the basis of the Einstein-Stokes
equation. Specifically, the proportionality of
the radius and the viscosity of the fluid
allows quantifiable Brownian motion in the
1um particle, but with the 3um particle the
force of gravity becomes quantifiable
relative to diffusive effects. The laser trap
was used to quantify the motion of the
beads within a liquid medium.

Diffusion Coefficient of Small

Polystyrene Beads
A buffer solution was prepared using the
buffer solution recipe xviii and 1m
Polyspherex Polystyrene Microspheres, a
slide was made and sealed with Maxi-Cure

Figure 3: Apparatus Diagram

Child & Thomas 4

superglue, hardened with Insta-Set. The

buffer was designed to minimize clumping
of polystyrene beads in the medium. Figure
4 demonstrates the construction of a slide.
After adding oil to the top of the coverslip
due to the use of an oil-immersion objective,
the slide was placed in the sample holder.
The objective lens was then set closer to
the sample holder to be touching the oil on
the slide.

Figure 4: Slide Construction

With the camera set level with the dichroic
mirror and the center of the microscope
objective lens, facing towards the light
source, the camera zoom was adjusted until
the beads were visible on an external
monitor. The dichroic mirror filters light
based on the wavelength. Specifically, light
at the lasers wavelength of 632 nm is
reflected, while higher wavelengths are able
to pass through the lens. This allows the
camera to be aligned with the path of the
laser as it enters the objective. The focus
was then adjusted by moving the objective
lens using the control on the stage controller.
Once the beads were focused on the
monitor, two minutes of footage was taken
using the digital camera. To allow for the
conversion of pixel measurements to
measurements in meters, the height of the

field of view was determined by locating a

bead at the top of the screen, recording the
y location from the stage controller, moving
the stage until the bead was at the bottom
of the field of view and then recording the
second y location. This physical distance
could then be correlated to the field height
in pixels.
The video was imported using video
manipulation software, and was then cut
into 10-second slices. This was done given
the limit on the computers memory. During
each step, care was taken to maintain the
frame rate and video resolution. Each slice
was then converted to 8-bit greyscale and
opened individually in ImageJ xix , a videoprocessing platform that breaks videos
down into static frames and allows photo
manipulation on a per-frame basis.
Greyscale was chosen because color was
not a necessary data point in our analysis,
and because converting videos to greyscale
lowered memory usage while editing. The
program was used to quantify the random
motion of the beads by measuring the
change in the pixel location of the beads as
a function of time, determined from the
change in the number of frames.
For each video clip, at least one bead was
identified that was in focus and its position
was recorded as a pixel coordinate. The clip
was then advanced 50 frames, at a known
frame rate of 30 frames per second, and the
pixel location of the bead center was again
recorded as a pixel coordinate. This process
was repeated for the duration of the clip, but
was ended early if the bead went out of
focus or the bead left the frame. The same
steps were taken for beads in each of the
clip slices until data was obtained for 100
changes in position for beads over 50-frame
intervals.

Child & Thomas 5

Using the measurement of the number of

pixels per distance in millimeters, each set
of pixel coordinates was converted to a set
of physical coordinates. Changes in position
were calculated by subtracting x and y
coordinate positions of the bead in one
frame from the x and y coordinates of the
bead 50 frames later. With knowledge of a
frame rate of 30 frames per second and
advancement of 50 frames between
measurements, the diffusion coefficient (D)
was determined using Equation 4, where
x was the change in x position of the bead,
y was the change in y position of the bead
and t was the change in time between
measurements.
D=<x2 + y2>/(4t)

(Equation 4)

A theoretical prediction was generated from

the Einstein-Stokes equation (Equation 5),
where kB was the scientifically accepted
value for Boltzmanns constant, T was the
air temperature of the room, was the
dynamic viscosity of waterxx , and R was the
radius of the bead size given on the Product
Data Sheet.
D= kBT/(6R)

(Equation 5)

Data for the 3m bead trials was obtained

automatically on a frame-by-frame basis by
using the Multitracker plug-in procedure
outlined in the lab manual. This procedure
computationally
returned
the
pixel
coordinates for a selected bead during each
frame of the entire clip.

Evaluation
constant

of

Boltzmanns

Using a similar procedure as for the 1m

beads, the motion of 3m beads provided
data for the evaluation of Boltzmanns
constant. Instead of using the manual
location of the beads within each frame of
the clips taken, the Multitracker plug-in for
ImageJ was again utilized. The average
velocity of the beads in the y direction was
determined from the displacement of each
bead over a set time period, in this case,
one frame. Again, care was taken to ensure
that the footage maintained a frame rate of
30 frames per second. The net force (Fnet)
on the bead was calculated from Equation 6,
where bead was the density of the bead from
the Polymer Data Sheet and water was the
density of water.
Fnet = (Bead Volume) x (bead - water) x
(acceleration of gravity)
(Equation 6)

The drag coefficient (Cd) was calculated by

dividing the net force on each bead by the
velocity of the bead. The diffusion
coefficient was calculated using only motion
in the x direction. Therefore, Equation 4
leads to Equation 7.
D=<x2>/(2t)

(Equation 7)

The Einstein relation was used to calculate

the Boltzmann constant for 3m beads
(Equation 8).
D = k BT / C d

(Equation 8)

With the introduction of the laser, the

second dichroic mirror the polarizer and a
focusing lens, a trap was created , as
shown in Figure 1. Again using the
properties of the dichroic mirror, the view of

Child & Thomas 6

the laser in the objective was filtered based

on its wavelength, thus allowing for
unobstructed video capture of the trapped
bead.

Laser Trapping
Slides of both stationary and mobile 3m
beads were observed in the presence of the
laser trap. Since the beads were able to
move in three dimensions and therefore
towards and away from the objective lens,
the microscope was focused at the depth of
each bead targeted. The chosen beads
were brought close to the focus of the laser
and observed to determine whether the
beads were drawn into the trap. Once a
bead was trapped, the slide was slowly
moved to determine whether the trap was
strong enough to control the position of the
bead as it travelled through the buffer
solution.

Human Blood Cell Trapping

A donors finger was cleaned with an
alcohol wipe and pricked using a 20G 1
Precision Glide, sterile needle. Two drops of
blood were mixed with 1ml of the buffer
solution used previously. A coverslip was
cut to of its original side and two slivers of
tape were placed along two opposite edges.
The coverslip was then stuck to a clean
slide and a piece of the coverslip paper was
placed between the coverslip and the slide.
Two drops of the blood-buffer solution were
injected onto the paper and allowed to
permeate under the coverslip. Once the
entire area under the coverslip was covered
with solution, the paper was removed and
the edge of the coverslip was glued and
hardened as before.
A drop of oil was placed on the face of the
slide and it was then mounted so that its
contents could be observed with the
objective lens. Blood cells were observed
on the slide and exposed to the laser. A
video both with and without the dichroic
mirror showed how the motion of the cells
changed in the presence or absence of the
laser.

Child & Thomas 7

Results
Diffusion Coefficient of Polystyrene Beads
Based upon the changes in position, velocities and diffusion coefficients found for each 1m
bead during each frame, average values over the entirety of the clip slices were calculated.
Table 1 shows these values along with the standard deviations based upon both manual and
Multitracker ascertainment of the beads positions.
<x> (nm)

<y> (nm)

<Vx > (nm/s)

Manual

43.352
60,939
25.510
(638.140)
(567.640)
(379.165)
Multitracker
-0.848
0.166 (92.200) -25.431
(124.046)
(3721.384)
Table 1: Motion of 1m beads ( in parenthesis)

<Vy > (nm/s)

<D> (nm2/s)
1.09
x
105
5
(2.88 x 10 )
1.79
x
105
(0.0602)

35.860
(337.299)
4.982
(2765.990)

Based upon the changes in position, velocities and diffusion coefficients in the x direction found
for each bead during each frame, average values over the entirety of the clip slices were
calculated. Table 2 shows these values along with the standard deviations.

Bead

<x> (nm)

<y> (nm)

<Vx > (nm/s)

3m Trial
Slice 1
3m Trial
Slice 2
3m Trial
Slice 3
3m Trial
Slice 4
3m Trial 2

1 -0.723
9.481
-21.682
(154.175)
(171.565)
(4625.243)
1 6.951
0.909
208.542
(148.835)
(172.273)
(4465.035)
1 -12.598
30.135
-377.952
(159.000)
(186.492)
(4769.869)
1 -11.422
-3.777
-342.647
(176.178)
(197.546)
(5285.349)
-2.213
7.542
-66.396
(170.441)
(198.806)
(5113.228)
Table 1: Motion of 3m beads ( in parenthesis)

<Vy > (nm/s)

-284.425
(5146.943)
27.274
(5168.176)
904.049
(5594.769)
-113.317
(5926.382)
226.282
(5964.194)

<D> (nm2/s)
355382.956
(566260.431)
331866.426
(486328.761)
379802.962
(697916,109)
465422.423
(704089.122)
435435.747
(924143.075)

Evaluation of Boltzmanns Constant

The average diffusion coefficient in the x direction for each trial was weighted based upon the
number of frames in the clip from which the diffusion coefficient was calculated, as shown in
Table 2. For example, Trial 1 Slice 1 consisted of 305 frames out of the 2155 total frames
analyzed. Therefore, the diffusion coefficient counted for approximately 14 percent (305/2155)
of the weighted diffusion coefficient.

Child & Thomas 8

Fram <x>
es
(nm)

<y>
(nm)

Trial 1 Slice 1

305

-0.723

-9.481

154.175

171.565

Trial 1 Slice 2

294

-11.422

0.909

148.835

172.273

Trial 1 Slice 3

215

-12.598

30.135

158.996

186.492

Trial Slice 4

221

-11.422

-3.777

176.178

197.546

Trial 2

1120 -2.213

7.543

170.441

198.806

Weighted

<Vx>(nm/ <Vy>
s)
(nm/s)

<Dx>
(nm2/s)

-21.682

-284.425 355382.95
6
208.542 27.274
331866.42
6
-377.951 904.049 379802.96
2
-342.648 -113.318 465422.42
2
-66.396
226.282 435435.74
7
-81.972
159.643 407500.95
5

Table 2: Weighted average of diffusion of 3m beads

The weighted diffusion coefficient was used in the calculation of Boltzmanns constant, based
upon the data shown in Table 3.

Diameter (um)

Bead Density (g/ml)

1.06

Water Density (g/ml)

Fnet (Newtons)

8.31265 x10-15

Cd (kg/s)

5.207 x 10-08

T (Kelvin)

296.75

Calculated kB (m2kg/(s2K))

7.15032 x 10-23

Accepted kB (m2kg/(s2K))

1.38065 x 10-23

Table 3: Calculation of Boltzmanns Constant

Trapped Bead
Table 4 shows the differences in average velocities in both the x and y directions as well as the
diffusion coefficient in the x direction as compared between a trapped and an untrapped 3m
bead.

Child & Thomas 9

Bead

<x> (nm)

Trapped 3m -0.422
(11/29)
(52.526)

<y> (nm)

<Vx> (nm/s)

<Vy> (nm/s)

<Dx> (nm2/s)

0.167
(45.540)

12.672
(1575.791)

5.025
(1366.208)

36181.706
(39518.143)

-81.972

159.643

407500.955

Untrapped 3m

Table 4: Comparison of trapped and untrapped bad motion

Blood Cell Trapping

A comparison of the characteristics of the motion of a trapped and an untrapped blood cell are
shown in Table 5.
Blood Cell

<x> (nm)

<y>
(nm)

<Vx
> <Vy
> <Dx>
(nm/s)
(nm/s)
(nm2/s)

Untrapped

6.561 (286.506)

-9.067
(102.53
4)

196.849
(8595.1
79)

271.996
(3076.0
18)

692573.
016
(600742
2.000)

Trapped

-3.108 (195.326)

-1.816
(237.91
1)

-93.229
(5859.7
88)

-54.492
(7137.3
33)

708872.
453
(655987
2.790)

Table 5: Comparison of trapped and untrapped blood cell.

Discussion
Brownian Motion
The results clearly show that observations
of the 1m bead are likely Brownian. This is
due to the non-deterministic motion, small
displacements,
and
high
standard
deviations of the numbers.

Diffusion
Coefficient
Polystyrene Bead

of

The
respective
diffusion
coefficients
calculated for the 1m and 3m beads
show that the effects of gravity were

negligible for the 1m bead when

comparing its average x and y displacement.
In the 3m bead, the quantifiable increase
in average y displacement relative to
average x displacement means that gravity
may be reasonably assumed as the
introduced force, compared to the 1m
bead.

Boltzmanns Constant
Our calculation of Boltzmanns constant is
surprisingly accurate at 317% greater than
the accepted value. However, the precision
was low based on the high standard
deviations of the data.

Child & Thomas 10

Increasing the precision of our answer

request two things. First, more data will
decrease error margins. Second, a major
source of error is the radius of the beads.
Understanding
the
manufacturing
tolerances of the beads will give a better
understanding of the precision of the
calculation.
Finally, the most precise way to calculate
Boltzmanns constant based on available
resources would be with a macroscopic
system. Calculating the gas constant based
on
relating
pressure,
volume,
and
temperature of a known molar quantity of
gas would give us the quantity equal to
Boltzmanns constant times Avogadros
number.

Optical Trapping
Limited experimental success was achieved
due to difficulties in obtaining a proper
alignment. This limited available data for
optically-trapped
beads.
In
addition,
difficulty was encountered with identifying a
correct
bead
dilution.
An
overlyconcentrated bead solution caused the
aggregation of multiple beads in a single
trap. We observed as many as seven beads
being held by a single trap. This data was
unusable for a quantification of bead
movement due the presence of external
forces.
However, data shows that trapping was
achieved in some capacity for the 3um bead
and blood cell. Further experimentation is
required to determine the strength of the
optical trap. Specifically, moving the bead
with the trap may be used to determine the
theoretical spring constant of the trap.

Our results show limited success in

capturing a blood cell based. This is
because, while average displacement
decreased under trapping, the data lacks
the precision to assert that the change is
relevant.
In the future, obtaining more data to
increase the precision of these readings
could prove more conclusively that the cell
was trapped. In addition, conducting
experiments based on moving the trapped
cell to demonstrate the Hookean nature of
an optical trap would be beneficial to
quantifying the force on the bead.
Further research could also be done by
binding polystyrene beads to the cell
membranes of blood cells, thus allowing
mechanical properties of the membrane to
be determined. In addition, attempts to trap
cell organelles could allow for study of the
viscosity and movement of the structures in
the cell. Specifically, studies of these
organelles during mitosis could further
understanding of the event based on the
ability to isolate or move organelles during
mitotic stages.

Error
Because Multitracker was not used initially
and the locations of the centers of the
beads
were
performed
manually,
discrepancies
between
the
diffusion
coefficients calculated compared to actuality
can stem from error in the locations.
Alignment was a major source of error.
Ensuring that the laser was perfectly aligned
to shine through the objective, with its focus
lying between the slide and cover slip was a
major difficulty.

Biological applications
Child & Thomas 11

The diameter of the bead was not known

precisely.
Specifically,
manufacturing
tolerances are an unknown and probable
cause of error. Therefore, some of the error
in the calculation of Boltzmanns constant
can be attributed to this discrepancy, since
the equation used to calculate the constant
includes the drag coefficient, which is based
upon the beads volume.
Because only a few seconds of data were
collected for the trapped and untrapped
blood cell, the results obtained were
imprecise. However, we note that the
average delta Y does indicate some effect
by gravity that was negated by the trapping
- so the cell had gravitational effects
between the 1um and 3um beads.
Operator error is a possible factor in the use
of Multitracker. Specifically, it was infeasible
to check every single frame of a given video
to ensure a homogeneous cell shape of
reasonable form, thus causing possible
error in the identification of an objects
boundaries.
The specific type of cell trapped in blood
was unknown. Knowledge of its type would
provide a better understanding of its
properties and shape.
One final significant source of error was that
we were working with a three-dimensional
system, but were attempting to quantify it in
two dimensions. Hence, there was motion in
the third dimension that was not accounted
for. This caused problems with focus as the
bead came in and out of view. In addition, it
affected the accuracy of our calculation of
Boltzmanns Constant.

Child & Thomas 12

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Child & Thomas 13