Experiment N◦ 5: Optical Confinement

Group N◦ 36 : Tristan Henchoz, Andrew O’Sullivan

March 2, 2021 Simicic Dunja

Introduction
During this experiment the motion of bead in a solution of water were studied. Initially the
motion of the beads will behave like a Brownian motion since they interact with smaller particles
(water). While in a second time, the motion of the beads will studied with confinement. This
optical confinement will be created by a laser creating a potential well which traps the beads.
Optical confinement it often used in biology in order to manipulate microscopic samples without
damaging them.

1 Theory
1.1 Brownian Motion
A particle has a Brownian motion when it interacts with a fluid, where the molecules are a
lot smaller then the particle. The numerous collisions with the molecules of the fluid create a
random motion, called Brownian motion. The Langevin equation [1], as written in equation (1),
describes this motion :

mẍ = −λẋ − kx + ξ (1)

where −λẋ is the force corresponding to viscous friction, −kx is a spring force associated with
the laser and ξ is a stochastic force (thermal agitation).
By multiplying equation (1) by x and successively taking the average of the gaussian noise
ξ, yield the following equation (2).
D E D E D E D E
m x = 2m x − λ x − 2k x˙2 + 2 hξxi
¨2 ˙2 ˙2 (2)
In the non confined case (k = 0), by taking the definition of the gaussian noise hξxi = 0
and the equipartition theorem (see equation (8) below), equation (2) can be rewritten as such :
D E D E
m x¨2 = 4kB T − λ x˙2 (3)
m
By taking an integration constant A and by taking λ
 t,it is possible to obtain
D E 4k T
 4kB T
˙ B
2
x = =⇒ x2 ∝ t (4)
λ λ
The quantity hx2 i, called mean squared displacement (MSD), indicated how much a particle
moves compared to it’s initial position, Numerically the MSD can be computed as seen in the
following equation [2]:
N −t/δt
1 X
MSD(∆t) = (x(iδt + ∆t) − x(iδt))2 (5)
N − t/δt i=1
where ∆t is the time step taken for the measurements of the position, δt is the time step
between frames in the video and N is the total number of measured positions.
The mean square displacement as function of the time step between measured positions ∆t
should behave linearly as such :
MSD(∆t) = 2dD∆t (6)

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where d is the number of dimensions of space used for the calculation of MSD and D [m2 /s] is
the displacement coefficient which will be determined experimentally.
The theoretical value for the displacement can be computed with the following formula :
kB T
D= (7)
6πrη
where kB is the Boltzmann constant, T is the temperature, r is the radius of the beads and η
is the dynamic viscosity of the medium. Ceci permet de réécire (3) comme h~x2 i ∝ 4Dt.

1.2 Confined motion

For the laser used here, the potential is approximated by a parabolic shape and by using the
Equipartition Theorem (Viriel) it is possible the find the trap stiffness k [Nm−1 ]:
 
1 2 2 1 2 ∂H
V = k(x + y ) = kr and r = kB T −→ khr2 i = kB T (8)
2 2 ∂r
The probability of finding a molecule in a potential V (x) in a volume dx in thermal equi-
librium is described in Boltzmann statistics by the following equation:
 
V (x)
p(x)dx = C exp − (9)
kB T
This equation can be inverted in order to get the shape of the potential as a function of the
probability p(x):

V (x) = −kB T ln(p(x)) + kB T ln(C) (10)

By injecting the potential V (x) inside equation (9), a Gaussian distribution (centred around
x = 0) is obtained:
 
k 2
p(x)dx = C exp − x (11)
2kB T
The same distribution in the y direction will appear. Therefore by using a Gaussian fit on
a histogram of the position in x and y directions of the beads, the variance σ 2 of the fit will be
extracted and the trap stiffness k for each direction can be obtained :
kB T
k= (12)
σ2
The trap stiffness for each direction should be the same.

2 Description of the experimental setup

2.1 Preparation of the samples
Before studying the motions of the small beads inside the water, the samples have to be pre-
pared. Beads of different sizes will be diluted inside water at different ratios in order for the
beads to behave as a Brownian motion. This is done with the use of a pipette by diluting
several times, until the desired dissolution percentage is reached. Once that is done, one or
two stripes of double scotch, depending on the size of the beads, are placed on either side of a
glass sample, then a cover slip is placed on top of the glass and by pressing it will stick to the
double scotch. Then the solution of beads is inserted between the two surfaces with the use of
the pipette and the whole system is sealed with nail polish and left to dry. This is redone for
every size of beads and every experimental day.

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2.2 Observation of the motion of the beads
When the samples are ready to be studied, a drop of immersion oil in placed over the sample,
before placing the whole on a translation stage above the illumination source as seen in figure 1,
which illustrates the experimental setup. The positioning of the translation stage and the
objective above are adjusted so that the immersion oil adheres to both the cover slip and the
objective and secondly in order to get a sharp image of the motion of the beads thanks to the
camera.

Figure 1: Experimental setup with the following elements (1 - illumination source, 2 - sample,
3 - objective, 4 - eyepiece lens, 5 - camera. Optical tweezer: 6 - laser, 7 - neutral filter, 8,9 -
mirrors, 10,11 - lenses, 12 - dichroic mirror, 13 - shortpass filter) [2]

2.3 Recording the motion of the beads

After the sample has been put in place, the motion of a bead can be observed with the camera
and recorded. Once the video of the motion of a bead is recorded, it is time to analyse the
motion. The software used allows the user the get the coordinates (xpixel (t), ypixel (t)) of the
bead as a function of time. Since the coordinates are in pixels a conversion ratio has to be
calculated. This is done be moving by a well defined distance the translation stage and observing
the difference in pixels on the camera.

2.4 Analysis of the motion

For Brownian motion, the movement (x(t), y(t)) is observed in order to verify that the bead
recognition software worked and to visualise Brownian motion. Once this is done for several
beads of different sizes, there respective MSD are calculated. By applying a linear regression for
the MSD, the experimental displacement coefficient Dexp can be calculated by using equation (6)
and compared with the theoretical one, which is calculated by using equation (7).
For the confined motion, the movement (x(t), y(t)) is observed in order to verify that the
bead recognition software worked and visualise the confined motion, where the particle should
remain within a certain distance from the centre of the laser. Once than is done the average
distance squared hr2 i is calculated in order to get a first estimation of the trap stiffness k with
equation (8), which will be called kequipartition . A second way to estimate the trap stiffness will
then be implemented with the use of a histogram. A Gaussian fit will be applied on a histogram
of the x(t) positions and the y(t) positions, yielding the values : kx and ky . A third method to

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calculate k, can be used by using a parabolic fit on the points obtained by equation (10), where
the probabilities are taken from the histogram. These three estimation of the trap stiffness will
be calculated for different bead sizes and different laser intensities I [W] and compared.
Finally a χ2 test will be done in order to verify the parabolic shape of the potential.

3 Results
3.1 Pixel-distance conversion
As can be seen in figure 2, the translation stage was moved manually in the x direction and
the bead moved accordingly on the screen. The same was done in the y direction.

(a) Position of translation stage x = 4.01 mm, (b) Position of translation stage x = 4.07 mm,
center of bead xpixel = 280 ± 10 center of bead xpixel = 1180 ± 10

Figure 2: Conversion pixel to distance (900 ± 14 pixels ⇐⇒ 0.060 ± 0.005 mm)

By doing the calculations for x and y we get that :

1.0 mm ⇐⇒ 15000 ± 200 pixels (13)

3.2 Motion of non-confined beads

Measurements for the motion of bead of different sizes were done. By measuring the mean
square displacement as defined in equation (5) and applying a linear regression, the displacement
coefficient D can be computed. Several measurements were done for each bead size and the
average displacement coefficient D was taken.
In the following figures the motion of a bead is described. Figure 3 shows the Brownian
motion of the bead along the x-axis and y-axis for two sizes of beads. These were the sizes
which displayed significant motion on the camera unlike the larger sizes.

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 (a) Bead of size r = 1.93 ± 0.05 µm (b) Bead of size r = 3.00 ± 0.07 µm

Figure 3: Motion in space of the beads

Figure 4 shows the mean square displacement for a particular bead for two different sizes.
As can be seen, the linear relation which was predicted is clear and with a linear regression the
displacement coefficient D for these bead can be calculated. Since measurements for several
beads were done for each size, the average value for each bead size is taken, giving therefore a
more accurate measurement for Dexp , which are given in table 1.

(a) Bead of size r = 1.93 ± 0.05 µm (b) Bead of size r = 3.00 ± 0.07 µm

Figure 4: Motion in space of the beads

By using the dynamic viscosity of the medium (water) ηwater = 8.94 · 10−4 Pa · s [3] at
ambient temperature T = 293 K, the theoretical value for D can be obtained also

r [µm] Dexp [m2 /s] Dtheo

Sample 1 10.1 ± 0.71 MSD failed (2.38 ± 0.17) · 10−14
Sample 2 5.93 ± 0.29 (4.4 ± 0.9) · 10−14 (4.0 ± 0.2) · 10−14
Sample 3 3.00 ± 0.07 (1.1 ± 0.4) · 10−13 (8.0 ± 0.2) · 10−14
Sample 3 1.93 ± 0.05 (1.6 ± 0.3) · 10−13 (1.24 ± 0.03) · 10−13

Table 1: Comparison between theoretical values Dtheo and experimental values Dexp calculated
through linear regression

3.3 Measurement of the trap stiffness k of confined beads

For the second part a laser of wavelength λ = 658 nm is used to confine the beads. Since the
beads have a certain energy and the potential should have a parabolic shape, the beads should

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remain inside a certain circle around the centre of the laser.
Unfortunately the beads of size 5.93 ± 0.29 µm didn’t show any signs of movement and the
smaller beads of size 0.99±0.03 µm which visually illustrated very well the confinement since the
free motion is quite significant, couldn’t be analysed. The software didn’t seem to recognise the
beads despite changing the contrast. Therefore measurements were only successfully analysed
for the beads of size 1.93 ± 0.05 µm and of size 3.00 ± 0.07 µm.
Measurements were done for different intensities of the laser going from the free motion to
the maximum power of 20.7 ± 0.1 mW. Since the objective is the calculate the trap stiffness k
for different laser powers and bead size, three methods are used.
As can be seen in figures 5a and 5b which is illustrate the different calculated values of k as
a function of the laser intensity I, no clear relation can be established. The values for intensity
of 8.5 ± 0.1 mW have much larger k values.

(a) Bead size r = 1.93 ± 0.05 µm (b) Bead size r = 3.0 ± 0.07 µm

Figure 5: Trap stiffness k as a function of intensity

A potential explanation for these different results can be given in figure 6, where the motion
of all beads of size 3.00 ± 0.07 µm for three intensities are compared. As can be seen the lower
intensity (8.5 ± 0.1mW) has a smaller radius of movement. This appear to indicate that the
bead is more stable for these lower intensities. This explains potentially the higher trap stiffness
for I = 8.5 ± 0.1 mW.

Figure 6: Confined motion for different intensities

A potential explanation for the large differences for the three different methods of computing
k can be given in figure 7. In this figure, the computed potential obtained by using equation (10)
on the data obtained from all measured beads of size r = 3.00 ± 0.07 µm and for intensity 12.4

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mW is shown in blue. The probability values p are taken from the histogram and C is a
constant that normalised the probability distribution. The fit obtained from the gaussian of
the histogram and the parabolic fit applied on the points in blue are also shown. These fits are
quite different. This can be explained by the fact that the gaussian fit gives greater importance
to the positions in the centre, while the parabolic fit gives a greater importance (relative to the
gaussian fit) to the points more on the outside. The figure 7 shows how V (r) computed via the
gaussian fit, matches the points at the centre very well, while the parabolic less so. This figure
appears to indicate that the region where the gaussian fit worked has a parabolic potential in
a small radius, while for larger distances other forces intervene, thus explaining in part at least
the difference between the two fits.

Figure 7: Confined motion for laser power of 12.4 ± 0.1mW

3.4 Verifying parabolic potential V

In the previous parts, a Gaussian fit was applied on the histogram in order to find the trap
stiffness. However the question should be asked whether the Gaussian fit is the correct approx-
imation or should another model be applied. The objective here is therefore to verify whether
the histogram has a Gaussian shape and therefore whether the the potential is parabolic.
In order to do this approximation, a χ2 test is done by comparing the expected amount of
frames in which the bead is in a certain space interval [x, x + dx] with the measured amount of
frames.
The χ2 test was done on all the measurements. The histograms fitted better the Gaussian
curve for beads of size 3.00 ± 0.07 µm then the smaller size, which was expected since the
smaller beads had a tendency to escaped from the potential. More surprisingly however, the
best results were obtained for average intensities ≈ 9 mW and not the maximum intensities.
Perhaps since the stronger intensities of laser had a smaller confinement area, getting the bead
in this confinement area was less successful.
In figure 8 the bead with the best χ2 is shown. As can be seen the p-values are high which
means it’s very unlikely that the potential isn’t parabolic. Usually a hypothesis (here parabolic
potential) is rejected if the p-value is below 5%, which is not the case.

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 (a) Histogram in x (p-value = 0.15) (b) Histogram in y (p-value = 0.94)

Figure 8: Gaussian fit with very good p-values (P ower = 8.6 ± 0.1 mW)

In order to demonstrate more visually the parabolic potential, V is calculated from the
probability distribution as given by equation (10). The potentials V (x) and V (y) obtained are
shown in figure 9 were V (x) and V (y) have a parabolic shape and have a similar curvature
(second derivative). This figure confirms p
therefore since both have a similar curvature, that the
potential is a function of the radius r = x2 + y 2 .

Figure 9: Parabolic potential in x and y directions

4 Discussion
The Brownian motion gave good linear regression for the mean square displacement and Dexp
and Dtheo were comparable for all sizes, except for the largest were the software had problems
tracking the bead.
For the confined motion, despite numerous tests, many samples were not analysed either
because there wasn’t any movement for the larger beads, or because the software failed to track
the smaller beads. Therefore comparison between different sizes became impossible. Only the
beads of sizes r = 3.00 ± 0.07 µm and r = 1.93 ± 0.05µm were analysed. Despite this optical
confinement was observed as can be seen by comparing figure 3 with figure 6. The difference
with the Brownian motion is quite apparent, since the beads are moving in a much smaller
area. The difference was the most apparent with the beads of size r = 0.99 ± 0.03 µm but
software didn’t track their motion.
The strength of the laser k as a function of the intensity didn’t yield any clear results. The
measurements for I = 8.5 ± 0.1mW gave stronger trap stiffness but this seems to come from the

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fact that the beads were more stable. For the other intensities a general tendency for increasing
k as I increases seems to appear at least for the beads of size r = 3.00 ± 0.07 µm. However
the different results obtained via the three methods (Equipartition theorem, gaussian fit and
parabolic fit) doesn’t allow a clear answer.
The parabolic shape of the potential was verified successfully. However the results were a
lot better for lower intensities as seen in figure 9 compared to figure 7 where a stronger intensity
was used. This again appears to confirm that the beads seemed more stable in traps of weaker
intensity. Therefore it is possible to affirm that the potential was parabolic, however other
factors meant that this was seldom observed apart with the larger beads and for the lower
intensities.

Conclusion
The measurements for the Brownian motion worked well for different sizes and the experimental
and theoretical values of the displacement coefficient matched for the different sizes. The
confinement was less successful. Software problems meant that the smaller beads weren’t
tracked and no conclusive relation between the trap stiffness and the intensity of the laser was
reached. However the parabolic shape of the potential created by the laser was accurately
verified at least for weaker intensities.
Nevertheless despite a lack of conclusive measurements for the optical confinement, this
trapping technique was observed and the differences could be seen with the naked eye on the
camera, in particular for smaller beads. This confirms the practical usefulness in biology or
other fields of optical trapping in order to move microscopic samples without damaging them
through direct contact.

References
[1] Branimir Luki, Sylvia Jeney, Zeljko Sviben, Andrzej J. Kulik, Ernst-Ludwig Florin and
László Forró, PHYSICAL REVIEW E 76, published july 2007

[2] EPFL, "TP3 : Confinement Optique", https://sb.epfl.ch/files/content/sites/fsb/

files/sph/TP3/notices/ConfinementOptique.pdf, consulted on the 6th april

[3] WIKIPEDIA, "Viscosity", https://en.wikipedia.org/wiki/Viscosity, consulted on

the 26th april

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Appendix, Data
A Error calculations
2 !1/2
 ∆T 2  T
  
kB 
∆Dtheo =   +  ∆T 
 r2 (14)
6πη  r  

Linear regression yi = axi :

P
xi y i
a = Pi 2 (15)
i xi
Average slope for N measurements for the mean square displacement :
N rP
1 X (xi − x̄)2
ā = ai ; ∆ā = (16)
N i=1 N −1

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