1

Laser Optical Tweezers (LOT)

Prepare for this experiment by studying the described theory on optical trapping and the operation
principles of the setup and data analysis (section 1 to 3). In this experiment you will work with lasers.
Carefully study the section on laser safety. In the quiz you will be asked about all above aspects.
In this small research project you will study active vesicle transport with Laser Optical Tweezers. Before
you start experiments in living cells a thorough characterization and calibration of the optical tweezers is
important. Software and manuals are available here: \\campusmp\software\Practicum Software and
Manuals\Laser Optical Tweezers (LOT)

In the assignment you will find several literature questions. Select one of these questions to answer and
discuss in your experimental report.

Laser safety
Introduction
Lasers are coherent light sources. Light is radiated as a nearly parallel beam. This characteristic makes
lasers dangerous. Through a (convex) lens, such as the eye, the parallel light beam is focused into one
point. At that moment all the energy of the laser light is dissipated onto a small surface. The high energy
density of the light source may also be dangerous to the skin.

In order to estimate the danger of lasers to some extent, the following is a simple calculation of
an HeNe laser with a capacity of only 1 mW and a 1 mm2 beam diameter. By experimenting you
can easily determine that a laser beam with an intensity of 1 W/mm2 can be felt burning on the
skin of your finger. The 1 mW HeNe laser has an intensity of 1mW/mm2 on the skin, which
does not cause any noticeable heating of the skin. In principle, the eye is capable of focusing this
beam 5.105 times (diffraction limit) onto the retina. The intensity on the retina resulting from a
1 mW HeNe laser then becomes 500 W/mm2, which is much higher than the 1 W/mm2 that you
feel burning on your skin! If you do not move your eye and the beam focuses perfectly on the
retina, the laser beam of a 1 mW HeNe laser may cause a severe retinal burn. This damage is
irreversible.

A laser beam of only a 1 mW HeNe laser may already cause permanent retinal damage
If you look directly into the beam!
Therefore avoid any eye contact with laser beams!

Laser classification system

In terms of radiation danger, lasers are subdivided into four classes. The following is a summary of the
classification system.
Remark: different classifications apply to different wavelength lasers and for lasers with different pulse
duration and beam characteristics.

Class 1 lasers
In this category a distinction is made between lasers of a very low power or built-in laser systems that are
intrinsically safe (Class 1), and lasers of a very low power where the beam diameter has been made very
large or very divergent and which will only constitute a potential danger when using optics (Class 1M). The
upper power limit for continuous lasers in the visible area is only 0.5 W.
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Class 2 lasers
In this category a distinction is made between visible, low-power lasers that are safe as long as the
exposure to the eye is less than 0.25 s (blinking reflex), and visible, low- power lasers where the beam
diameter has been made very large or very divergent that, in the combination with optics (for example
lenses), may be dangerous if the eye is exposed to the beam for less than 0.25 s (Class 2M). Visible and
continuous lasers ranging from 0.5 W to 1 mW are classified as Class 2.

Class 3 lasers
In this category a distinction is made between low-power lasers that are considered safe if used with care
(Class 3R) and the medium-power lasers that may cause damage if the eye is exposed to the beam (Class
3B), also with regard to specular reflecting surfaces such as glass or metal (watches, rings, jewelry, etc.).
The upper power limit for visible and continuous lasers is 5 mW in Class 3R and 0.5 W in Class 3B.

Class 4 lasers
High-power lasers that may cause eye injury or skin damage, also in the case of specular or diffuse
reflection. This class of lasers may also involve a fire risk.

The impact of laser radiation on living tissue

Laser radiation is a specific type of visible (VIS), ultraviolet (UV) or infrared radiation (IR). It’s impact is
mainly of a thermal nature. The high power density of this type of radiation may cause effects that are
barely noticeable for ‘normal’ UV, VIS and IR radiation. Very intense pulses, for example, can deposit that
many energy in a cell, that the fluid within the cell evaporates and the cell explodes due to increased
pressure. Non-thermal effects may also occur, such as photochemical reactions leading to the generation
of free radicals and subsequent tissue damage. The thermal impact, however, is dominant. It is mainly the
skin and eyes that are at great risk of being damaged.

The skin
The most pronounced effect of laser beams on the skin may be the occurrence of burns, which can
sometimes be really severe. Furthermore, UV lasers may induce skin cancer. The skin, however, can endure
higher levels of energy than the eyes, because there is no beam concentration (focusing effect of the eye
lens).

The eyes
The eye is the most sensitive organ considering laser radiation. The lens concentrates the already intense
beam of visible or near-infrared rays by a factor of 100 to 5.105. This may cau se serious damage,
particularly if the beam hits the macula lutea, the yellowish central area of the retina that contains the
photoreceptors of the optic nerves. For example, a 40 mW HeNe laser produces approximately a 10
kW/cm2 radiation intensity on the retina. This radiation intensity instantly causes thermal damage of the
retina, similar to boiling egg white.

Figure 1: Various forms of eye damage to the retina of a monkey,

caused by a Nd:YAG laser. The white spots in the centre were
caused by thermal charge (burning spots). Craters and
hemorrhages (the dark spots) appear when higher power levels
are used. The damage is permanent. Source: [4]
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The radiation level that can be regarded as the theoretical limit between 'safe' and 'possibly safe' is
indicated by the MPE (Maximum Permissible Exposure). The International Commission on Non-Ionising
Radiation Protection (ICNIRP) has determined the MPE levels for the eyes and skin for lasers that have a
wavelength ranging from 180 nm to 1 mm and a radiation period ranging from 10-13 s to 30000 s.
It should be noted that the MPE standard does not define a clear-cut limit below which laser radiation may
be considered 'safe', because these MPE standards were mainly determined through vivisection. Section
Three of the User's guide of the International Standard IEC 60825-1 of 1993, including amendments 1:1997
and 2:2001 that were drawn up by the International Electrotechnical Committee, can be consulted for
additional information about MPE levels and how to calculate them for particular laser types. The NEN-EN
207 and NEN-EN 208 standards can be consulted for the requirements with which laser safety eyewear should
comply.

Regulations for working with lasers

The following instructions for safely working with lasers are general rules that you always should comply with.
These, however, should be regarded as guidelines and they most definitely do not exclude every form of risk
in all instances where lasers are being used.
 Unintentional reflections should be avoided.
 Unnecessary specular reflection of surfaces should be avoided.
 No reflecting rings, necklaces, watches etc. should be worn when working in the laser laboratory.
 All optic components should be mounted in a fixed position to a table (in relation to the laser source)
or to another surface, so as to avoid sudden changes in the direction of the laser beam.
 Laser beams must never run at eye level through the room, but should preferably run along an
optical table.
 Whenever and wherever possible, beam blockers and black screens should be used to block
undesired beam reflections beyond the optical table.
 Alignment work should preferably be carried out with a reduced-power laser beam.
 If possible, alignment work should be carried out in a well-lit area (small eye pupils reduce the risk of
eye injury)
 Workplaces should be set up in such a way that laser beams can never run at eye level.

The most common causes of accidents in research labs (as obtained from actual case histories) are:
(1) not wearing appropriate safety goggles,
(2) not reducing power for alignment procedures, or unintended power increase,
(3) stray beams left uncontained by beam stops or other barriers.

Are you safe?

The Accessible Emission Limit (AEL)
The primary measurement of a laser’s hazard potential is the Accessible Emission Limit (AEL), which
defines the maximum total power of radiation that can be emitted from a laser of a particular class.
Assuming a linear additive effect for radiation absorbed by the eye, the minimum irradiance known to
cause a biological effect is converted into a power level for the length of time defined by a given class.
As AELs are mainly used for classification of a laser, they are not immediately useful to a user who wants
to know if his/her particular setup is safe. However, you can use the classification scheme to help you
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make very simple decisions. For instance, if you always keep your power level below that required of a
class II device, you can be assured that accidental exposure to the beam will not be hazardous to you. For
visible lasers, this means keeping the average power below 1 mW for startup or alignment procedures.

The Maximum Permissible Exposure (MPE)

The most useful number in laser safety calculations is the Maximum Permissible Exposure (MPE). This is
the maximum irradiance or radiant exposure that may be incident upon the eye (or the skin) without
causing biological damage. The MPE is related to the AEL by the limiting aperture of the eye, which is itself
a function of wavelength and exposure time. To make things simple, the MPE is tabulated separately (see
table 2).

The basic method for evaluating the safety of your specific laser system is to calculate the maximum
irradiance that an unprotected eye might experience while the system is operating in a particular manner,
and check whether it is less than the MPE.

Useful Approximations
For each laser in use, we are interested in identifying the maximum safe energy that may be incident upon
the eye. This energy is often a rather complicated function of wavelength and the duration of exposure.
In some places a single value suffices for a broad range of conditions. In other cases, notably those of
visible (400-700 nm) and near-infrared (700-1400 nm) radiation during ordinary time-periods of exposure,
the MPE should be calculated on a case-by-case basis. For these purposes, it is worth using the following
assumptions:
- Limiting time of inadvertent exposure to visible radiation = 0.25 s (the blink reflex)
- Limiting time of inadvertent exposure to UV (180-400 nm) or NIR radiation = 10 s (natural eye
motion)
- Limiting time of exposure for intentional viewing = 100 s (i.e. during laser alignment)
You might think the estimated exposure time of 100 s for a procedure such as alignment is ridiculous.
However, even if you are wise enough to use indirect methods such as burn paper or power meters to
align your beam, it is entirely possible that you will look in the same direction for 100 s during an alignment
procedure. The 100 s exposure time includes the possibility of unbeknownst exposure to a stray beam
while performing an operation on your laser system. Once you have determined MPE relevant to a
particular application, you must calculate the level of incident radiation at your eye under a variety of
circumstances. Important parameters for such calculations are the optical path length from laser output
to the eye, the beam diameter and divergence, the limiting aperture of the eye, whether the beam is
viewed as a point or extended source, and whether a reflection is considered specular or diffuse.

Important Conventions
Before calculating actual numbers, you have to be sure you have the right starting values and units. The
three common numbers found in laser manuals are the beam diameter, the beam divergence, and the
radiant energy or radiant power. These values appear in safety calculations, where they have the units of
cm, radians, J and W respectively. The central quantities in a laser safety calculation however, are the
radiant exposure H (measured in J/cm2) and the irradiance E (measured in W/cm2).

The MPE is determined by averaging the incident power of the beam over an area defined by the limiting
aperture of the eye. For visible light this is the diameter of a fully dilated pupil, which is 7 mm. For non-
visible radiation other limiting apertures are defined as listed in Table 1.
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Table 1: limiting apertures per spectral region and exposure time

Spectral Region Period of Exposure (s) Aperture Diameter (mm)

-9
180 to 400 nm 10 to 0.25 1.0
4 3.5
0.25 to 3 × 10
-9 4
400 to 1400 mm 10 to 3 × 10 7.0
-9
1400 nm to 100 mm 10 to 0.25 1.0
4 3.5
10 to 3 × 10
-9 4
100 to 1000 mm 10 to 3 × 10 11.0

Example of MPE calculation

The MPE for accidental exposure to a visible laser (400-700 nm) is calculated by taking an exposure time of
0,25 s, the blink reflex. From Table 2 we obtain:

𝑀𝑃𝐸 = 1,8𝑡 /
× 10 = 0,64𝑚𝐽/𝑐𝑚 , expressed in the radiant exposure
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Table 2: Maximum permissible exposure (MPE) for C6 = 1 at the cornea for exposure to laser (from NEN-EN-IEC 60825-1)
Exposure time t
Wave s
-
length 10–13 10–11 10–9 10–7 1,8  5  1 10–3 10 10 2 103
to to to to 10–5 10–5 to to to to

10– 10– 10–7 1,8  to to 1 10 10 3 104
nm
11 9 10–5 5 10–5 1  0 2 3
10–3
180 to 302,5 30 J m –2
3 1010 W m –
2 Photochemical hazard d
302,5 to 315 (t > C2 J m–2
T 1 ) C2
J m –2
Thermal hazardd
(t T1)
315 to 400 C J m – C1 J m–2 104 J m –2
400 to 450 1,5 10–4 J m –2 2,7 104 t 0,75 J m –2 5 10–3 J m –2 18 t 0,75 J m–2 100 J m –2
C3 W m –2
450 to 500 100 C3 J m–2
and c
10 W m -2
500 to 700 10 W m -2
700 to 1 050 1,5 10–4 C4 J m –2 2,7 104 t 0,75 C4 J m – 5 10–3 C4 J m –2 18 t 0,75 C4 J m–2
2 10 C4 C7 W m –
1 050 to 1 400 1,5 10–3 C7 J m –2 2,7 105 t 0,75 C7 J m – 5 10–2 C7 J m–2 90 t 0,75 C7 J m –2 2
2
1 400 to 1 500 1012 W m –2 103 J m–2 5 600 t 0,25 J m –2
1 000 W m –2
1 500 to 1 800 1013 W m –2 104 J m –2
1 800 to 2 600 1012 W m –2 103 J m–2 5 600 t 0,25 J m –2

2 600 to 106 1011 W m –2 100 J m –2 5 600 t 0,25 J m –2

a Fora)
correction factors andfactors
units, see
For correction andTable 10see
units,
Table A.2
b) The MPEs for exposure durations below 10–9 s and for wavelengths less than 400 nm and greater than 1 400 nm have been derived by
calculating the equivalent irradiance from the radiant exposure limits at 10–9 s. The MPEs for exposure durations below 10–13 s are set to
be equal to the equivalent irradiance values of the MPEs at 10–13 s.
c) In the wavelength range between 450 nm and 500 nm, dual limits apply and the exposure must not exceed either limit applicable.
d) For repetitively pulsed UV lasers neither limit should be exceeded
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Table 3: Correction factors and breakpoints for use in MPE evaluations (from NEN-EN-IEC 60825-1)
Parameter Spectral region
nm
C1 = 5,6 103 t 0,25 180 to 400

T1 = 100,8(–295) 10–15 s 302,5 to 315

C 2 = 30 180 to 302,5

C2 = 10 0,2(–295) 302,5 to 315

T2 = 10 10 [(– )/98,5]

min s 400 to 1 400

T2 = 10 s for  < 1,5 mrad 400 to 1 400

T2 = 100 s for  > 100 mrad 400 to 1 400

C3 = 1,0 400 to 450

C3 = 10 0,02(–450) 450 to 600

C4 = 10 0,002( –700) 700 to 1 050

C4 = 5 1 050 to 1 400

C5 = N–1/4 a 400 to 10 6

C6 = 1 180 to 400 and 1 400 to 10 6

C6 = 1 for    mi nb 400 to 1 400

C6 = / min for  min <    max b 400 to 1 400

C6 =  max / min = 66,7 for  >  max b,c 400 to 1 400

C7 = 1 700 to 1 150

C7 = 10 0,018(–1 150) 1 150 to 1 200

C7 = 8 1 200 to 1 400

 min = 1,5 mrad

 max = 100 mrad

N is the number of pulses contained within the applicable duration (8.3 f) and Clause
A.3).

NOTE 1 There is only limited evidence about effects for exposures of less than 10–9 s for
wavelengths less than 400 nm and greater than 1 400 nm. The AELs for these emission
durations and wavelengths have been derived by calculating the equivalent radiant power
or irradiance from the radiant power or radiant exposure applying at 10–9 s for wavelengths
less than 400 nm and greater than 1 400 nm.
NOTE 2 See Table 11 for aperture stops and Table A.4 for limiting apertures.
NOTE 3 In the formulae in Tables 4 to 9 and in these notes, the wavelength must be expressed
in nanometres, the emission duration t must be expressed in seconds and  must be
expressed in milliradians.
NOTE 4 For emission durations which fall at the cell border values (for instance 10 s) in
Tables 4 to 9, the lower limit applies. Where the symbol “<” is used, this means less than
or equal to.
a C5 is only applicable to pulse durations shorter than 0,25 s.
b C6 is only applicable to pulsed lasers and to CW lasers for thermal retinal limits.
C The maximum limiting angle of acceptance  th shall be equal to  max (but see 8.4 d)).
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1 Introduction
An optical trap or ‘optical tweezers’ is a device that can apply and measure forces as small as piconewtons.
A highly focused light beam, often a laser hence the name laser optical tweezers, is obtained in a
microscope. This focused light beam can manipulate micron sized dielectric objects, e.g. small polystyrene
or glass beads (spherical particles) or even organelles in a living cell.
Optical tweezers (also known as an optical trap, laser trap, or gradient force trap) was first reported by
Arthur Ashkin of Bell Labs in 1970 [1]. Most recent applications for optical tweezers have been in the field
of biophysics where they can be used to measure the piconewton forces exerted by molecular motors
such as those found in DNA polymerase or of a molecular motor moving a vesicle along an actin fiber. In
order to accurately measure these molecular motor forces we need to have a well-calibrated instrument.
Calibration for optical tweezers means knowing what the trap stiffness and trap force are for a laser beam
at certain intensity. Much like a spring, the approximate force that the laser beam applies to an object is
proportional to the object’s displacement from the center of the beam.

2 Theory
Physics of optical trapping
The most straightforward mechanism to understand the physics of trapping is to consider the change in
momentum of light that is scattered and refracted by the dielectric material, for example a polystyrene
bead. Any change in the direction of light imparts momentum to the bead. This mechanism based on ray-
optics holds for objects much larger in diameter than the wavelength of the laser (typically d>10).
A microscopic particle in a focused laser beam experiences forces in the axial and transverse dimensions
from two effects. The first is a “gradient force” that draws the object into the center of the trap in both
the transverse directions (perpendicular to the laser beam propagation) and the longitudinal direction.
The second force is due to scattering of photons off the object and creates a longitudinal force that pushes
the object in the direction of light propagation, which results in the trapping location in the center of the
laser beam but slightly past the focal point.

Figure 2: A ray diagram showing how the gradient force stabilizes the trap laterally. [5]

When a bead moves slightly away from the center, a net force is applied towards the center, making this
a stable equilibrium. In order to understand how the equilibrium is stable, it will help to consider how the
gradient force responds to displacement of a bead from the center. As seen in figure 2, the red region
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represents the “waist" of the laser at its focus point, with the laser passing upward. The blue ball is the
bead, and the dark red arrows (1) and (2) represent light rays whose thicknesses correspond to their
intensities (note that the beam is brightest at its center). In case (a), with the particle slightly to the left of
center, the two rays refract through the particle and bend inwards. The reactionary force vectors, F1 and
F2, of each ray on the bead are shown as black arrows. Because ray (2) is more intense (and thus carries
more momentum) than ray (1), the net force on the bead is to the right. Thus, a perturbation to the left
causes a rightward-directed force back towards the trap's center. In case (b) the particle is centered
laterally in the beam and will not be pushed left or right. The net gradient force is downward, which is
balanced by an upward scattering force (not shown) due to reflection of some of the light. When the
reflectivity of the bead is too high, the scattering force will actually push the bead out of the trap.

This ray-optics approach to describe the principle of optical trapping holds for trapped objects much larger
than the laser wavelength. Ray-optics are not valid for objects smaller than the wavelength and the
phenomena should be described by Rayleigh scattering. You may consult chapter 4 of the user guide of
the setup for more information on this topic.

Trap Stiffness
The optical trap can be viewed as a spring as shown in Fig. 3, where the force pulling the object back to
the trapping position is proportional to the distance from the trap center.

Figure 3: Optical trap acts on the particle as a spring with spring constant (or stiffness) k. The negative sign
simply indicates that the force acts to restore the bead to the center of the trap (i.e., if the bead moves in
the +x direction, the force is in the –x direction). [6]

The strength or stiffness of the optical trap depends on a number of parameters. The focusing power of
the objective lens and contrast in refractive index of the dispersive medium and the trapped object
determine the maximum attainable force. A more strongly focused laser beam results in higher gradient
forces. Larger particles are also more easily trapped than small ones. Finally, the laser power of course
determines the trap stiffness. For a given system, with a fixed focus and particle size and material, the
latter is the only parameter to adjust the trap stiffness.

To be able to measure forces on trapped particles, the trap stiffness needs to be determined. To this end
we can study the displacement of a trapped particle from the trap center under influence of force. Since
Work = Force  distance, but force changes with distance, the potential energy at a given x is the area
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under the curve of Force vs. distance. Note that the force in this case is the force required to
counterbalance the spring force in order to pull the sphere out of the trap, or
Fx  k x (1)

Particles suspended in liquid exhibit random movement due to collisions with the moving molecules
(thermal motion) that make up the liquid. This phenomenon is known as Brownian motion. The
equipartition theorem states that a molecule in thermal equilibrium has an average kinetic energy for
each degree of freedom given by:
1
E k BT ,
2
where kB is Boltzmann’s constant, T is the temperature in Kelvin, and E is the energy in Joule. A bead in an
optical trap will also show Brownian motion although now the motion is restricted by the trapping force.
A particle trapped in a harmonic potential well (i.e. a mass on a spring) has an energy:
1
Ep  k  x2  (3)
2
where <x2> is the time-averaged square of the bead’s horizontal displacement from the center of the trap.
This is the statistical variance in the position of the bead due to Brownian motion. Assuming that the
movement of the trapped bead is due only to thermal fluctuations, we can set the kinetic energy equal to
the potential energy of the trap:
𝑘 < 𝑥 >= 𝑘 𝑇 (4)
By measuring the Brownian motion of a trapped bead, we can experimentally determine the stiffness of
an optical trap without having to know any information about the fluid viscosity or geometry of the
trapped particle.

Brownian motion of a freely diffusing particle

Particles in solution that are not restricted in their motion exhibit free Brownian motion. Characteristic of
this motion is the random walk of the particle through the fluid. The motion of the particle is described
by the Einstein relation:

6𝜋𝜂𝑟𝐷 = 𝑘 𝑇 , (5)

in which  is the dynamic viscosity of the fluid medium, r is the particle radius and D is the diffusion
coefficient. From this relation you can calculate the theoretical diffusion coefficient for a bead in solution.
The diffusion coefficient is generally expressed in m2/s.

In an experiment you can determine the diffusion D for a freely moving particle in solution by analyzing
the particle trajectory. The trajectory of a diffusing bead can be described by the Einstein-Smoluchowsky
relation:

< (𝑟⃗(𝑡 + 𝜏) − 𝑟⃗(𝑡)) >= 2𝑑𝐷𝜏 , (6)

where 𝑟⃗(𝑡) is the trajectory of the bead, t the time,  the time interval between observed positions and d
the dimension. The brackets <> mean that the average is taken over many time intervals. This mean
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squared displacement (MSD) can be calculated for different time intervals. For unrestricted Brownian
motion the plot of the MSD against the time results in a straight line with slope 2dD.

Literature question 1: The Einstein-Smoluchowsky relation, as described

above, is appropriate in the case than no external forces are present.
Although this is an appropriate starting point for describing diffusion, it is
possible to derive expressions for different types of diffusion than free
diffusion. For instance, forced diffusion will result in more rapid diffusion
than linear, where restricted diffusion will limit the MSD, see figure A.
When a polystyrene bead is placed in an optical trap, it experiences a
harmonic potential. Find literature that describes diffusion in a harmonic Figure A: Forced (super-), free (normal) and restricted
(sub-) diffusion (image by J. Krieger [Wikipedia user])
potential and briefly elaborate on your findings.

Stokes Drag
Another method to determine the trap stiffness and strength uses the viscosity of the liquid. In this
method the sample is moved at a constant speed and the viscous drag on a sphere as it is held still in the
trap results in a drag force on the sphere. This drag force is called the Stokes drag force:

𝐹 = 6𝜋𝜂𝑟𝑣

The drag force Fd depends on the velocity vf of the fluid flowing along the bead, the bead radius and the
viscosity of the fluid. This dynamic viscosity is 1.10-3 Pa.s for water.

The configuration used for this method is shown schematically in Fig. 4, where the sphere is held in the
trap while the fluid surrounding the sphere is moved to the left. The drag force experienced by the sphere,
Fd, must be balanced by the trap force, Ft, for the sphere to stay in the trap. As the drag force is increased,
the sphere will move more off center so that the trapping force increases via Eq. (1), maintaining force
balance.
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Figure 4: Schematic diagram of drag force experiment. The sphere is held in the trap while the surrounding
fluid is moved to the left at speed vf. The equilibrium displacement of the sphere, x, is used to relate the
drag force to the trap force. Source: open-source webpage.

From this force balance, the relation between the fluid velocity and the bead displacement from the trap
center can be obtained. When the viscosity and bead size are known the trap stiffness can be determined
from this relation.

Literature question 2: The application of an optical trap is very prominent in biological physics research.
The length scales at which optical tweezers can be used, are typical for many fundamental kinetic
processes in (plant) cells. An example of these processes it the motion of protein motors which account
for active transport within the cell, see section 4.
In order to gain detailed insight of the fundamental processes happening in the cell, which are typically
discrete mechanical movements, the spatial resolution of the optical trap must be small enough to detect
these small steps individually instead of measuring a collection of consecutive steps at once. Find
appropriate literature that describes what limits the spatial resolution of the optical trap and briefly
describe what physical phenomena are most influential in limiting the spatial resolution and the typical
length scales associated with the processes.
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3 Experimental setup
Setup and installation
The laser optical tweezers setup contains the following main components (consult the manual for more
details):
1. Trapping laser and controller. Be careful with this laser, never look into the beam or reflections
of it!

2. Laser focusing and alignment system. Shielded for safety.

3. Objective lens to make focused laser spot onto sample.

3
7 2 1
4 5 1
6

Figure 1: [7]
4. Power supply LED
5. Motorized translational sample stage.
6. Controllers for x-y translation.
7. Micrometer for z-focussing.
8. Camera.

To operate the setup you need software to control the laser and sample positioning stages (APT control
software) and software to operate the camera (uc480 software package). Download the installation from
the LOT folder (\\campusmp\software\Practicum Software and Manuals\Laser Optical Tweezers (LOT)).
Carefully follow the instructions as listed in the manual of the setup on pages 34 up to 39. Pay special
attention to the settings of the actuator motors (page 39); do not use the values enlisted in figure 35 at
page 40.
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After installation of the laser controller software (APT user), check the settings of the laser power (this is
not always set correctly). Set the maximum current to 110 mA. A laser power of 75 mA is generally more
than enough to trap a particle.

The setup is pre-aligned. If you think something might not be okay in the alignment, always notice the
practicum staff. Do not try to change anything in the alignment, this will most certainly result in loss of
the trapping ability. Even for an experienced operator it takes a few hours to do a full alignment.

Samples and procedures

You will start your experiments with the observation of the Brownian motion of polystyrene beads in
solution and their behavior in the optical trap. Samples containing beads can be obtained from the
practicum staff. There are 1 micron diameter beads available. A few microliters of a sample should be
suspended onto a microscope slide which can be positioned on the sample positioning table.

IMPORTANT: be very careful not to touch the sample with the objective! This may cause severe damage
to the objective. Also do not touch this objective with your hands or fingers. When (re)placing a sample
always move the sample stage to the lowest position. When focusing pay attention to the distance
between objective and sample. The working distance of the objective as only a few hundred microns. This
means you always work at close range between objective and sample. When moving the sample stage
vertically always first make sure that you move in the right direction.
For your initial experiments to image the beads and to find the trap, follow the procedure described in
section 7.2 (p. 44) of the user guide.

The sample is imaged with the camera. To be able to record distances and velocities in the experiments
you need to convert pixels to real distances in the sample. For this purpose a calibration grid is available
(to be obtained from the practicum staff). Another way to calibrate the camera is to move the stage over
a fixed distance with the x-y microcontrollers. The distances and speeds of the microcontrollers are
calibrated (see user guide).

Data analysis
All data consist in movies recorded with the camera. The data are best analyzed in Matlab. There are
scripts available to load the movies into Matlab; you can select regions of interest in the movies, this
greatly reduces data processing time. There are also scripts available to determine the position of beads
in the movies and to trace their trajectory. Some script will be provided with some initial routines to
analyze your data. Where necessary you have to adapt these scripts to process your data.
IMPORTANT: do the analysis simultaneously with your experiments, you need the analysis to be able to
judge if you have measured something useful. If you process your data later you might find that they are
all useless.

4 Experiments
Brownian motion of a free moving particle
Record the Brownian motion of 1 m diameter beads in solution. When you start first make sure you have
a number of beads nicely in focus, so you obtain a clear image from the camera. Record a movie and
analyze this with the Matlab script ‘ImageAnalysisFramework’. You need to record a long enough movie
(many frames) to be able to do an accurate analysis. Trace the trajectory of several beads (you can do this
in one movie simultaneously) and determine the diffusion coefficient of the beads from these trajectories.
 15

Use the method of mean squared displacement for this analysis. The user guide provides more
information for this procedure (p. 49-50).
Hint: sometimes you observe large jumps in the trajectories. This is of course not related to Brownian
motion. You can remove these parts and analyze the ‘good looking’ parts separately. Argue why this is
allowed.
Repeat this experiment a few times sizes to obtain good statistics and reliable data. Compare your
measured values with the theoretical value.

Trap stiffness
The stiffness of the optical trap is the important parameter that determines the amount of force the trap
can apply to an object. Quantify the trap stiffness with the method of variance analyses and by applying
a fluid drag force. Compare the results of the two methods. To calibrate the trap's spring constant versus
laser supply current you will need to perform each of the following measurements at several laser powers.
You must use at least three, but preferably five different powers.

Variance of a trapped bead

In your experiments consider the following notes:
- Record the movement of a trapped bead long enough to get enough statistics in your data.
- When processing your data, make use of a region of interest in the recorded frames just around
the trapped bead. This greatly reduces processing time.
- Check your results (the reconstructed trace) if the analysis worked out well. Try varying the profile
used to fit the bead position.
Do this experiment for the 1 m beads.
Perform several measurements for different laser powers. This works best using the same trapped bead;
start at high power and record the bead’s motion. Then reduce the power stepwise and record the motion
for each step. Make a plot of the variance as function of the laser power. Obtain from these plots the trap
stiffness. There is a relation between the variance and the trap stiffness you can use to fit the results.

Analyze these data also with the mean squared displacement method like you did for free Brownian
motion. Discuss the results in comparison with those obtained for a freely diffusing particle. Plot the MSD
data as function of time. Make sure you incorporate large enough time intervals. What do you observe?
Explain this result. What property of the optical trap can you derive from this?

Drag force method (this method is optional, since it takes quit some time)
In your experiments consider the following notes (next to the ones mentioned above):
- Move the sample with the programmable stage controller (use the sequencer tab, in the
controller window), this will induce a fluid flow around the trapped bead.
- There is a change that the laser focus changes when you move the sample too much, due to some
skewness in the sample stage. A solution could be to move the sample up-and-down for a short
range.
- Make sure that the programmed speed is indeed reached. How can you check this? High velocities
need some time to be reached, depending on the acceleration. Check the operation instructions
(Manual Servo Motor Driver TDC001, e.g. p. 59 Fig. E2) of the device to see how this works.
In this experiment you observe the displacement from the trap center as function of applied drag force.
Do this for different laser powers. Think over what the best experimental procedure would be.
Determine the trap stiffness for the different laser powers from the obtained results of displacement as
function of drag force.
 16

Another parameter you can simultaneously extract from these measurements is the maximum trap force
for each laser power. The drag force at which the particle escapes from the trap is equal to the maximum
trap force.

Strength of the Actin-Myosin Molecular Motor and Intracellular Transport of Vesicles in Onion Cells
(challenge experiment)
Our goal in this section is to analyze the force exerted by Myosin to move the granules in the onion cell.
These small granules (or vesicles) are about 1 micron in diameter.

In this part of the experiment, you will observe the motion of particles inside a living cell. Cells transport
food, waste, information, etc. in membrane-bound vesicles, which are visible under a light microscope.
Though diffusion is an important mechanism, it is too slow and random for long distance transport and
directing materials where they are most needed, especially in larger cells. It is now understood that cells
have highly developed and intricate mechanisms for directed transport of materials.

Most motions within and of cells involve two components, a cytoskeletal fiber that serves as a track, and
a motor protein that does the work. The motor molecule uses energy from the hydrolysis of one ATP
molecule to bind to the fiber, bend to pull itself along the fiber, and release, all of which constitutes one
"step".

Cartoon of myosin motors pulling organelles along an actin filament. Source: [8]

Virtually all cell types exhibit directed intracellular transport, but some cell types are particularly suitable
for transport studies. We will use epidermal cells from onion bulbs. With some care, a single layer of cells
can be peeled off an inner section of the onion bulb and mounted flat on a slide.
 17

Onion cells in bright-field illumination.

Round object in each cell is the nucleus
Source: [3]

Vesicles in the cytoplasm of a plant

cell, as seen in dark-field. Source: [3]

In this experiment, we will be viewing the movement of vesicles within the cytoplasm of onion epidermal
cells, shown above as they appear in bright-field and dark-field microscopy. The layers you see in an onion
bulb develop into leaves when it sprouts. Both sides of the leaf are covered with an epidermis consisting
of brick-shaped cells, each with a cell wall and cell membrane on the outside. Most of the interior of the
cell is filled with a clear vacuole that functions in storage and in maintenance of hydrostatic pressure
essential to the stiffness of the plant (the difference between crisp lettuce and wilted lettuce). The
cytoplasm, containing all of the other cell contents, occurs in a thin layer between the cell membrane and
the vacuole, and in thin extensions through the vacuole called transvacuolar strands. It is within the
cytoplasm that you will be observing directed transport of vesicles by an actin-based mechanism. These
vesicles are spherical or rod-shaped organelles such as mitochondria, spherosomes, and peroxisomes
ranging in size from 0.5 to 3 microns. The diagram of an onion cell below shows the location of the cell
wall, cytoplasm and vesicles in a typical cell; you will not be able to see much of the endoplasmic reticulum
or the vacuole depicted because of their transparency. Under the microscope, you will notice the vesicles
are located just along the edges of the cell, or near the top and bottom surface if you focus up and down.
When you see a narrow band of moving vesicles in the center of the cell, it is located in a transvacuolar
strand, which may be a handy place to study motion.
 18

A 3D cross-section model of an onion epidermal cell, showing actin filaments and vesicles in the narrow
bands of cytoplasm within the cell. Source: [3]

In plant cells, vesicles are transported along actin fibers by myosin motor molecules. In some plant cells
and algal cells, a large-scale streaming motion of the cytoplasm is observed, logically called cytoplasmic
streaming. This bulk flow is believed to be caused by myosin motors pulling the extensive endoplasmic
reticulum along actin fibers lining the cell membrane. Many other vesicles are then dragged along with
the endoplasmic reticulum. Lodish and Berk, et al. provide a detailed explanation of this process and a
video of cytoplasmic streaming in the pond weed Elodea can be viewed here.

Literature question 3: In the experimental guide it is stated that the motor proteins, which transport
vesicles along the cytoskeletal fiber, uses ATP molecules as “fuel”. How these steps made by the motor
protein are made can be found worked out in literature. Kinesin is a protein that belongs to the class of
motor proteins. The different variants of myosin, which are forms of kinesin, are found in plant cells. The
‘roads’ on which these motor proteins move are the cytoskeletal fibers. These motor proteins in plant
cells can transport different types of materials in a cell actively. This means that the transport is not merely
done by diffusion.
Find appropriate literature describing the principles of the motion of motor proteins along the cytoskeletal
fiber. (Briefly explain how FIONA (fluorescence imaging with one nanometer accuracy) is used to measure
these steps.)
 19

The experiment
In your observations of vesicles in onion epidermal cells, you should distinguish between the random
Brownian motion of vesicles that are unattached (or at least not actively moving along) actin filaments,
the directed transport of vesicles by attached myosin motors, and possibly bulk flow of vesicles in
cytoplasmic streaming.
Obtain an onion cell monolayer on a slide. Note that the cell is large compared to the field of view of the
microscope. The onion cell sample is a monolayer of onion cells with a few drops of saline solution held
under a coverslip. Onion layers are separated by loosely attached monolayers of cells, and thus these
samples are readily prepared from a typical, ordinary onion.

Figure Typical onion cell with clearly visible vesicle pathways. Source: [9]

Spend some time observing the behavior of this system and recording your observations. (Use screen
capture images, video, and written narrative as appropriate to provide context to what is being observed.)
Identify the round, “hollow" vesicles, looking for one which is being transported at a steady speed along
a linear trajectory (the actin micro-fiber). Trap such a moving vesicle by moving it into the focused laser
beam (set at moderate to high power). Once the vesicle is trapped it cannot be transported anymore.
Now gradually decrease the laser power until the vesicle is able to move again along the actin fiber. Note
the point at which the actin-myosin motor is able to overcome the force of the optical trap. Use your prior
calibration of laser current versus trapping stiffness to determine the force required to stop the actin-
myosin motor. Repeat this measurement a sufficient number of times to quantify the uncertainty in the
stall force.

Making an onion slide

 Take one of the lower layers (activity depends somewhat on depth) and remove the lower
membrane using the forceps, this is similar to pulling off a sticker. The easier it peels off, the less
damage to the cells, so try several.
 The membrane is a single layer of cells which makes it particularly clean when viewing through a
microscope. It should appear translucent and should be relatively strong.
 Make a slide using this membrane
o Place a drop of saline solution onto a clean slide (don't use water)
o Place the membrane onto the drop of saline on the slide
o Drop another drop onto the onion and cover with a cover slip
 20

o Blot excess liquid using a paper towel

 Mount onto microscope
 Keep in mind that the lifetime of an onion slide is about 30-60 minutes before it dries out.
 Put the remainder part of the onion into a plastic baggy and put it into the refrigerator.

Observing cells and trapping vesicles

Scan the slide to find regions where cells aren't covered by air bubbles or badly damaged. Look for cells
with an intact nucleus and some non-random motions of small vesicles. Occasionally a preparation will
not show good motion and you should try another slide.

Examine patterns of motion within a cell. Some regions will show little activity, others will have large
numbers of vesicles in motion. To study internal transport along actin filaments it will be helpful to find
relatively isolated tracks along which small numbers of vesicles travel. Try trapping vesicles and see what
happens to the movement of other vesicles along the same track. How does the vesicle behave when it is
released? With a vesicle trapped, you can try moving it through the cytoplasm by moving the stage.

Here are some ideas for your investigation:

1. How do vesicles in active transport respond to manipulation by the trap? Does stopping and
releasing a vesicle result in resumed motion, motion in the opposite direction, or ceasing of
motion? What effect does trapping a vesicle have on other vesicles travelling the same route?
2. Calibrate the trap stiffness using a vesicle. Many of the vesicles are similar in size to the beads
you've used for calibration, but the vesicle's optical properties and the viscosity of the cytoplasm
differ from the beads in water you used. By trapping a vesicle that is showing Brownian motion
(not active transport), you could use the equipartition method to estimate stiffness. How does
this compare with values obtained with beads in water for similar laser powers?
3. Measure forces involved in active transport of vesicles. Stop a vesicle and then reduce laser power
until the vesicle can break free and try to estimate the force at this power. This may be tricky to
do. How do these measurements compare with the Stokes drag force calculated using vesicle size,
velocity, and cytoplasm viscosity?

Use your prior calibration of laser current versus trapping stiffness to determine the force
required to stop the actin-myosin motor. Repeat this measurement a sufficient number of times
to quantify the uncertainty in the stall force.

Literature question 4: In this experiment, the motions of vesicles along the cytoskeletal fibers is
studied. However, why these vesicles are being transported and what kind of vesicles are
transported to which part of the cell is not specified in the guide so far.
Find out, citing appropriate literature, what the most important mechanisms in the onion cell are
that rely on the active transport of vesicles along the cytoskeletal fibers. Is there a difference
between the transport function of active and passive vesicles?

5 References
1. A. Ashkin, Phys. Rev. Lett. 24, 156 (1970).
2. This text and experiment was adapted from the optical trapping experiment of MIT Berkeley,
published on mediawiki, 2012.
 21

3. Brownian Motion in Cells (BMC) (published on Advanced Lab

[http://experimentationlab.berkeley.edu])
4. (Photography courtesy of J. Zuclich, TASC Litton, TX, USA, Cite: Webb, C. E., & Jones, J. D. (Eds.).
(2004). Handbook of Laser Technology and Applications: Laser design and laser systems(Vol. 2).
CRC Press.)
5. Made by Wikipedia user: https://en.wikipedia.org/wiki/Optical_levitation
6. https://www.scind.org/1301/History/a-journey-from-optical-pressure-to-optical-tweezers.html.
7. ThorLabs User Guide EDU-OT1/M Portable Optical Tweezers
8. Shimmen, T., & Yokota, E. (2004). Cytoplasmic streaming in plants. Current opinion in cell
biology, 16(1), 68-72.
9. Image from Mazurenko and Porras, 2011.