Optical Tweezers

Nanotechnology Report
Shraddha Raghuram | Daria Roman

2nd Year Nanobiology Bachelor Programme, 2022

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Contents
1. Introduction…………………………………………………………………………………3

1.1 Theory and background information………………………………………………3

1.2 Implementation of the technique…………………………………………………..3

1.3 Calibration of the instrument………………………………………………………5

1.3.1 Confined Brownian method……………………………………………...5

1.3.2 Drag Force method………………………………………………………5

1.4 Advantages of Optical Tweezers…………………………………………………..5

1.5 Limitations of Optical Tweezers…………………………………………………..5

1.6 Active areas of research around Optical Tweezers………………………………...5

1.7 Applications of Optical Tweezers in Biology……………………………………...6

1.7.1 Mechanics and unfolding of DNA, protein, etc…………………………6

1.7.2 Molecular adhesion (e.g. cell adhesion by cadherins)..............................6

1.7.3 Forces and step size of motors (e.g. kinesin, DNA polymerase)..............6

1.7.4 Forces applied to measure cell mechanics (e.g. red blood cells)..............6

2. Trapping Beads……………………………………………………………………………...7

2.1 Experiment Description……………………………………………………………7

2.2 Results……………………………………………………………………………..7

2.3 Improvements……………………………………………………………………...8

3. Trapping Vesicles in a Plant Cell……………………………………………………………9

3.1 Experiment Description……………………………………………………………9

3.2 Results……………………………………………………………………………..9

3.3 Improvements…………………………………………………………………….12

3.4 Biological question related to onion vesicles that could be studied using the
technology of Optical Tweezers……………………………………………………..12

References……………………………………………………………………………………13

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 1. Introduction
1.1 Theory and background information
In order to understand the functional basis of optical tweezers, we first have to trace the story
2 2 2 2
back to Einstein’s famous Energy-Momentum relation: 𝐸 = 𝑝 𝑐 + (𝑚𝑐 ) . This equation
tells us that the total energy of a particle is related to its momentum (dependent on motion)
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and its mass. When we refer to a particle at rest, we obtain the well-known formula 𝐸 = 𝑚𝑐 .
In the case of light, which infamously has no mass, the general equation is simplified to
𝐸 = 𝑝𝑐.
A point worth noting is that when we discuss moving, massless particles that do not travel at
the speed of light, we can choose a reference frame along which the particle would be at rest,
invalidating the Energy-Momentum relation. Therefore, massless particles are constrained to
travel at the speed of light c, and fortunately, photons are the most notable possessors of this
quality. [1] The momentum of a photon is related to the wavelength of light and is denoted
ℎ −34
by the formula: 𝑝 = λ
, where h is the Planck constant (6. 63 · 10 𝐽 · 𝑠). The momentum
of a single photon is clearly insignificant, however when we consider the total momentum of
many identical photons (i.e. laser) acting on small particles, it becomes noticeable.
Arthur Askin , the inventor of optical tweezers, first managed to trap a particle by using high
powered lasers, and consequently, the property of light carrying momentum, in the late 1980s.
Twenty years later, in 2018, his invention was awarded the Nobel Prize in Physics.

1.2 Implementation of the technique

Optical tweezers use gradient forces from a highly focused laser beam to trap particles, in a
manner similar to tweezers. This is why they are also called single-beam gradient force-trap.
The mechanism integrates the principles of Ray optics and the Rayleigh regime. In cases
where the radius of the trapped particle is much larger than the wavelength of the trapping
light, the optical tweezer follows the principles of Ray optics. The change in direction of the
light as it passes through the bead causes a change in momentum. This change in momentum,
along with the forces exerted on the bead towards the laser focus, creates a 3D trap for the
bead. If the bead moves away from the laser focus, a net force acts on it returning it to the
focus (i.e., the trap), because the laser beams at the focus impart greater momentum to the
bead, than away from the focus, which causes a change in momentum. When the bead is at
the center of the beam, individual rays of light are refracting symmetrically, therefore there is
no net lateral force acting on the bead. Therefore, the axial force on the bead cancels out the
scattering force of the light and holds the bead in place. [2] When the radius of the bead is
much smaller than the wavelength of the trapping light, the optical tweezers follow the
principles of the Rayleigh regime. The laser has an electric field, which induces a dipole
moment in the bead, which is typically made out of a dielectric material. This dipole moment

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is directly proportional to the electric field. The energy of this system can be denoted by the
→ →
following relation: 𝑈 =− 𝑝 * 𝐸. This is directly proportional to the electric field intensity.
The force on the bead would then be the gradient of the energy, which in turn would be
proportional to the gradient of the electric field. This would cause the force to point towards
the region of the electric field that has maximum intensity. This would be the focus and thus a
trap is set for the bead by the optical tweezers. [3]

Figure 1: Optical bead illuminated by an unfocused laser, following the principles of

ray optics, i.e., when r>>λ. [4]

The optical tweezers setup consists of a laser beam that passes through a double lens system
that expands the beam, to facilitate the coverage of the high aperture objective (NA≥ 1. 2).
The requirement for a high aperture objective ensures that the incident laser rays are oblique.
The dichroic mirror, placed after the objective, allows the transmission of the microscope
illumination (LED) wavelength, while blocking the high powered laser beam wavelength,
which will consequently not reach the CCD camera (recording device). Additionally, a
condenser is used to create the trap in the focal plane of the objective. There is another single
lens system that acts as a position detector for the beam displacements. Finally, we note that
the wavelength of the optical laser is near the InfraRed region (800-1000 nm), this
wavelength of light is known to be minimally absorbed by water and proteins, and at the same
time makes the cell the most viable. [4]

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Figure 2: Typical Optical Tweezers setup showing the laser path [4]

1.3 Calibration of the instrument

To understand the calibration of the optical tweezers, we implement Hooke’s law into the
physics of this instrument. According to this, 𝐹𝑡𝑟𝑎𝑝 =− 𝑘𝑡𝑟𝑎𝑝𝑥. We use this law since, for
small displacements, we can approximate the bead to have a harmonic potential and thus
behave like a spring. There are two methods for calibrating this instrument:
● Confined Brownian method
● Drag force method [4]
1.3.1 Confined Brownian method
This method uses the equipartition theorem, according to which,
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1/2 * 𝑘𝐵𝑇 = 1/2 * 𝑘𝑡𝑟𝑎𝑝 < 𝑥 𝑏𝑑
> .
2
Here, < 𝑥 𝑏𝑑
> is measured experimentally, and using the equation above, 𝑘𝑡𝑟𝑎𝑝 is
calculated. [4]
1.3.2 Drag Force method
The viscous drag force produced by a moving spherical particle in fluid is given by
𝐹𝑑𝑟𝑎𝑔 = 6 * π * η * 𝑅 * 𝑣, where η is viscosity, R is the radius of the bead, and v is its
velocity. This drag force is equivalent to 𝐹𝑡𝑟𝑎𝑝. So we thus get
𝐹𝑑𝑟𝑎𝑔 = 6 * π * η * 𝑅 * 𝑣 = 𝑘𝑡𝑟𝑎𝑝 * 𝑥 . In optical tweezers, drag forces are produced by
the periodic motion of the stage while the beads are held in the trap. At each time point the

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position of the bead is measured, and inputed into the formula above. Using this algorithm,
we can calculate the value of 𝑘𝑡𝑟𝑎𝑝. [4]

1.4 Advantages of Optical Tweezers

They provide better control of the forces acting on the bead, as compared to other methods
like magnetic tweezers, for example. Optical tweezers also allow us to change the position of
the trap at high frequencies by moving the beam using deflectors or mirrors. We can also
create multiple traps using special techniques with optical tweezers.[5]

1.5 Limitations of Optical Tweezers

The disadvantages of this technique include the fact that the lens cannot focus the beam
smaller than half the wavelength of light due to the Rayleigh criterion. Another limitation is
the overheating of the instrument, resulting in unwanted exertion of a larger force on the bead
than required.[6] Sometimes, it is also difficult to trap particles that move with a high
velocity.

1.6 Active areas of research around Optical Tweezers

A research team at University of Technology Sydney has developed a method to control the
refractive properties and luminescence of the beads being visualized by the optical tweezers
by doping their crystals with rare-earth metal ions, and optimized the concentration of these
ions to achieve the trapping of the beads at a much using much less power and with much
higher efficiency. These optical tweezers also have a higher degree of sensitivity, and result in
much less overheating than experienced previously. [7]
Deviation of the focus of the laser due to overheating of the object can be fixed by using
temperature controlled objectives. The power can be stabilized to a greater level by
monitoring the laser intensity using a photodetector and a feedback loop to control the
intensity. Transmitting the laser through an optical fiber can also help improve the
stabilization. [8]

1.7 Applications of Optical Tweezers in Biology

1.7.1 Mechanics and unfolding of DNA, protein, etc.
When visualizing mechanics and unfolding of nanoparticles, we observe that we can design
the nanoparticles to bind with different assay geometries: surface tethering, glass micropipette
and dual-beam tweezers (dumbbell shaped). While designing this, we encounter several
surface issues, such as nonspecific adsorption, protein denaturation, among others. Some of
these issues can be resolved by incorporating blocking proteins like bovine serine albumin,
casein, etc. Another solution could be to separate the molecules from the surfaces by
incorporating an ovehanging stretch of DNA, a “handle”. We could form the attachment with
tags in the following way:

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 ● Incorporating ligand-receptor pairs such as biotin/streptavidin, antigen/antibody.
● Covalent attachment of tags to the beads and molecules.
● The specific binding of the bead and molecule then produces a well-defined geometry
between them with a bond strength that can withstand upto a 100 pN. [4]

1.7.2 Molecular adhesion (e.g. cell adhesion by cadherins)

This can be observed by the use of optical tweezers and understood theoretically by analyzing
the data obtained and making a plot of the force exerted on the nanoparticle versus the
velocity of the nanoparticle. [4]

1.7.3 Forces and step size of motors (e.g. kinesin, DNA polymerase)
Some examples of molecular motors are kinesin, proteasome and replisome. These can be
successfully studied with the help of optical tweezers. [4]

1.7.4 Forces applied to measure cell mechanics (e.g. red blood cells)
A bead of a red blood cell can be trapped using optical tweezers. This can help us
differentiate between a parasite-free red blood cell and a red blood cell infected by
plasmodium falciparum (malaria). This technique can also be used to probe fluid motion
around a swimming microorganism. Cell-cell interfaces can also be deflected using optical
tweezers. [4]

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 2. Trapping Beads
2.1 Experiment Description
Following the approximation of the harmonic potential trapping behavior of Optical
Tweezers, we can correlate the value of the spring constant with respect to the input laser
power.

To achieve our goal, we perform an experiment consisting of capturing movies of trapped

plastic beads at increasing laser powers (10 to 35 mW with a linearly spaced step of 5mW).
For sample preparation, we use 2 µm sized beads immersed in liquid. The methodology is
further summarized in the following steps:
1. On a glass slide, place two vertical double-sided tape strips in parallel. The spacing
between the two strips is approximately 1 cm.
2. Cover the thin strips with a square shaped tape, forming a small, encapsulated and
elongated chamber, in the middle of the two parallel tapes
3. Carefully pipette in the beads-solution which will fill the chamber through capillary
forces.
4. Seal the chamber using nail polish.

After achieving the right sample configuration, we will continue with the sample visualization
and beed trapping. A key requirement for the experiment is the use of proper equipment,
protective glasses, as the procedure involves manipulation of powerful lasers. Note that the
microscope used in the experiment has a high aperture oil-immersion objective. Oil
immersion is frequently used for high numerical aperture objectives because of the high
refractive index of the oil medium. Prior to placing the sample on the microscope stand, we
add a drop of oil between the glass slide and the objective. Once the microscope illumination
is switched on, we look for a bead in our sample. Calibration of the laser focus is checked. If
the laser is in proper focus, a butterfly shape appears in our field of view.

After clearly finding a bead in the sample, we switch the lasers on and off with increasing
power from 10mW to 25 mW in steps of 5 mW. Data is acquired by taking movies of the
trapped beads. Finally, we mention the importance of using protective glasses and the careful
handling of high power lasers.

2.2 Results
The spring constant of the trap is calculated by measuring the mean displacement of the bead
in the lateral projection, x and y direction, with respect to the center of mass. The provided
code is run for the increasing laser powers. The final results can be visualized in Figure 2. We

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 −7
observe a positive linear dependence between the laser power and the 𝑘𝑡𝑟𝑎𝑝 (𝑝𝑁/𝑛𝑚) * 10
The linear regression unweighted fit has a R squared value of 0,9723, which further validates
the linear dependency noticed. In our hookean model, the 𝑘𝑡𝑟𝑎𝑝 constant, which quantifies the
amount of force required to deform a harmonically oscillating spring, increases as the laser
power increases. This finding is indeed sensible as the trap becomes ‘stronger’ directly
proportional to the laser power applied.

Figure 3: Trendline of the data points, ktrap values are computed with respect to the center of
mass of the bead in (pN/nm), and plotted against linearly spaced Laser Power values (mW)

2.3 Improvements
Throughout the experiment, a number of aspects could have improved the final precision
outcome, lowering the noise (standard deviations) of the measured displacements in our time
lapse movies. The most significant issue encountered was achieving laser focus on our
sample. This is because we had to manually move a joystick placed on top of the laser
chambre, leading to disruptive mechanical interactions. Means of controlling the positioning
of the slide without interfering with the instrumentation, would solve this problem.
Additionally, we propose an increase of the beads concentration immersed in solution, as the
efficiency of our data acquisition was negatively influenced by the time taken to find a single
bead in the sample.
With regards to computing the results, we argue that a refinement of the code would be the
ability to append sequential k-trap values to compute the final graph. This advance would lead
to the complete automatization of the process, such that we would not have to manually
retrieve the k-trap values. We note that the code would have to firstly return an interface for
the input of the number of measurements taken, and the linearly spaced laser power values.

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 3. Trapping Vesicles in a Plant Cell
3.1 Experiment Description
Following the first experiment which aimed to introduce the basic procedure of trapping
immersed plastic beads, we move on to a more difficult task, that of trapping vesicles in the
cellular membrane of plant cells, particularly onion. The increased difficulty of the
experiment is derived from the higher velocity of the moving vesicles, as compared to the
previously used bead sample. In order for the experiment to function properly, we note the
time dependence, as the onion sample should not dry up for the vesicles to still be moving at
a biologically relevant rate.

The sample preparation protocol is fairly simple, similar to the steps described in the trapping
beads experiment. The onion is peeled and roughly cut into smaller pieces, out of which we
will extract a very thin, outer layer using tweezers. Next, the thin piece of tissue will be cut
into a smaller piece that is placed on top of the glass slide. The glass slide is coated with a
drop of water to ensure the hydration of the tissue. Finally, a cover slip is placed on top of the
sample and any water protruding out of the cover slip is wiped off.

The optical set-up is the same as the one used in the previous experiment, including the
oil-immersion objective. All the samples are visualized in order to select the one that has the
clearest, high velocity vesicles for further trapping. The quality of the recorded image is
determined by the object focus, which is changed such that the output shows sharp images of
the microtubules and the vesicles. The protective glasses are used throughout the active laser
periods. The calibration step and butterfly shape is checked before proceeding to the trapping
stage of the experiment.

3.2 Results
The selected onion epidermal tissue contained cells with a considerable amount of vesicles,
making the identification of the globular vascoules much faster in comparison to the low
concentration solution of plastic beads visualized before. Vesicles are moving constantly at a
very high velocity in the cell, making it extremely difficult for us to capture the vesicles in the
trap. Trapping the vesicles, hence took a lot of time and many unsuccessful tries before a
successful one. We also needed to increase the laser power to a high enough level such that
trapping would be possible without damaging the sample. Below you can see images of the
trapped vesicles visualized by the CCD camera of the optical tweezers.

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Figure 4: Image of the untrapped moving vesicles as observed on the screen when captured
by the CCD camera before illumination by the laser beam.

Figure 4: Image of a moving vesicle trapped by the optical tweezers, post the illumination by
the laser beam. The noise in the left corner was from dirt particles present on the surface of
the camera.

Figure 5: Image of another trapped vesicle captured by the CCD camera of the optical
tweezers.

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During the experiment, we observed that some vesicles were approaching a constant velocity,
linear track following the cytoskeleton of the cell, consisting of microtubules. This
observation strongly indicated that the dynamic mechanism of the vesicles could involve the
use of molecular motors, attached to microtubules. When the vesicles became trapped with
lower powered lasers, the velocity decreased. After gradually increasing the laser power, we
noticed that the vesicles would become displaced from the microtubules and their movement
would be completely impaired for a few seconds.

3.3 Improvements
The results of the experiment described above were based on visual, human observation,
making it highly qualitative. We suggest an improvement for making this experiment more
quantitative by implementing particle tracking or kymograph analyses on ImageJ to the
movie we have obtained. This will return data regarding the displacement of the particles at
various time points, and using this we can calculate the velocity of each particle. Subsequent
averaging, and applying it to the formula for drag force, we can obtain a quantitative value for
the average drag force exerted on the particle.

3.4 Biological question related to onion vesicles that could be studied using
the technology of Optical Tweezers
Performing these two experiments one after the other, we clearly noticed a similarity in the
globular structure and size of the particles and the epidermal cell vesicles. Therefore, a
biological question integrating both experiments would involve the approximation of the
vesicle structures observed in the epidermal onion cell as the plastic beads used in the first
experiment. The refractive index of the plastic beads and that of the transparent medium
inside the vesicles could as well be approximated as equal. Based on the calibration graph
showing the k-trap values on the y-axis and the laser power on the x-axis, we can imply the
same k-trap results for the trapping of the vesicles. Therefore, we could determine the force
required to significantly displace the vesicle from the microtubular linear track by measuring
the displacement and appending the k-trap value with respect to the laser power used. This
would lead to a better understanding of the force exerted by the molecular motors which we
observed in the second experiment.

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References:
[1] Light has no mass so it also has no energy according to Einstein, but how can sunlight
warm the earth without energy?
[2]
Lynn Paterson "Novel micromanipulation techniques in optical tweezers", (2003)
[3]
Bradshaw DS, Andrews DL (2017). "Manipulating particles with light: radiation and gradient
forces". European Journal of Physics. 38 (3): 034008. Bibcode:2017EJPh...38c4008B.
doi:10.1088/1361-6404/aa6050
[4]
Marie-Eve Aubin-Tam (2022) Slides on Optical Tweezers
[5]
Fazal, F. M., & Block, S. M. (2011). Optical tweezers study life under tension. Nature
photonics, 5, 318–321. https://doi.org/10.1038/nphoton.2011.100
[6]
"Next-generation" optical tweezers trap tightly without overheating.
[7]
Shan, X., Wang, F., Wang, D. et al. Optical tweezers beyond refractive index mismatch using
highly doped upconversion nanoparticles. Nat. Nanotechnol. 16, 531–537 (2021).
https://doi.org/10.1038/s41565-021-00852-0
[8]
Bustamante, C.J., Chemla, Y.R., Liu, S. et al. Optical tweezers in single-molecule biophysics.
Nat Rev Methods Primers 1, 25 (2021). https://doi.org/10.1038/s43586-021-00021-6

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