Construction and Use of Setup for Neuron Ablation in
C. elegans
Sam Meehan
Advisor: David Biron
April 5, 2010
Abstract
In have constructed an experimental laser setup that is used to perform the ablation of neurons in the C. elegans nematode. Here I describe the setup, its current
functionality, and preliminary results. I further describe difficulties encountered,
goals for the continuation of this project, and an experiment I plan to perform in the
coming weeks.
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Introduction
One of the inherent difficulties in the study of neuroscience is the extreme complexity of
the systems involved. However, the Caenorhabditis elegans nematode offers a case study in
which the circuitry of the nervous system is minimal to the point where it can be studied
in analogy with an electric circuit. To this extent, we have constructed and begun to use
a pulsed laser setup to perform sub-micrometer precision surgery on neurons within C.
elegans. By removing single processes from the neuronal circuitry of the worm, we hope
to identify certain neurons or regions of neurons responsible for specific activities of the
worm. In particular, I plan to use this technique to study thermotactic behavior, a sense
attributed to the AFD neuron in the pharynx region of the worm. However, in the future,
this setup will be used to study sleep-like behavior in the worm.
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Background
The practice of using femtosecond pulsed lasers to perform microscopic surgery was developed nearly two decades ago and has been fairly constant over the past fifteen years [1].
Applicable in a wide range of fields, the precision of the laser systems used allows for damage to be performed on the scale of a few microns. Furthermore, the techniques for using
pulsed laser systems with C. elegans has been developed in previous efforts [2] [3].
Although still a topic of research, the theory of how the surgical precision can be
obtained is well developed. In general, damage to biological material can be obtained
by ionization of the material. The amount of ionization, α, that occurs depends on a
parameter called the optical intensity defined as
E
(1)
Aτ
in which E is the pulse energy, A is transverse size of the beam, and t is the pulse time.
For single photon ionization, the amount of ionization scales linearlywith I. However, in a
phenomenon called multiphoton absorption, α scales as the number of photons, k, required
for that type of ionization. When a highly focused beam is incident upon material, all k
values for multiphoton absorption are possible (Figure 1). However, because of the power
law scaling of α, there is some threshold intensity Ith . For I > Ith , multiphoton absorption
is the dominant contributor to ionization and the biological material, composed primarily
of water in worms, is transparent by comparison to single photon ionization. Using this
effect, a laser beam can be focused to a spot such that the intensity is great enough that
damaging amount of ionization will occur only in a localized region similar to that in
Figure 2.
I=
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Setup
The setup constructed is shown in Figure 3 with a few key components described in more
detail.
The laser used is the Coherent Ultra pulsed laser system that works with a Ti:Sapphire
gain medium which we set to operate at 800nm for ablation. Inside the laser a modelocking cavity that pulses the laser at 80MHz. There are also additional optical elements
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Figure 1: Ionization absorption versus optical intensity (left) and schematic cross section of
microscope objective focusing laser beam to produce region of nonlinear absorption (right).
Ablation is performed using intensities with I > Ith in small region indicated. Courtesy of
Chung et al. [2].
Figure 2: Example of typical ionization region created by nonlinear absorption effects.
This was taken using 488 nm wavelength laser light. Image courtesy of Vogel et al. [6].
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Figure 3: Shown here is a schematic of the experimental setup for ablation.
that work to clean the beam of any continuous wavelength laser light prior to leaving.
Lastly, there is a group velocity dispersion pre-compensator system just prior to a pulse
leaving the laser. This is comprised of two prisms that can be moved to create a variable
length path through which the beam passes. This can be used to adjust the phases of the
different wavelengths within a given pulse prior to leaving the laser so as to precompensate
for any variation in dispersion effects due to other optical elements in the setup [5].
After leaving the laser, the pulses pass through an electro-optical modulator (EOM).
This device is composed of a birefringent crystal housed inside of a capacitor. The EOM
is rotated in the beam path such that when a high bias voltage is applied, the polarization
is perpendicular to that of the laser and the beam is stopped. However the EOM is
synced to the laser through discriminator and amplifier electronics such that the electric
field is turned off for a selected number of regularly spaced pulses. For these pulses, the
polarization of the crystal is such that the beam passes through with little attenuation.
Functionally, this works as an ultrafast shutter such that enough energy is deposited in
the worm in a short enough time to cause ablation but prevent other undesirable effects.
The pulses which pass through are then directed through a manually controlled mechanical shutter and some directive mirrors before entering the microscope. Inside the
microscope there is a dichroic mirror (Figure 4) that reflects the 800nm laser pulses but
passes light in the visible region in which the fluorescent imaging of the specimens is performed. This mirror directs both the imaging light and the laser pulses into the 60× water
objective which is used to both focus the image and collapse the pulse size to the submicron
scale necessary to perform ablation.
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Current Progress
My work during the course of the winter quarter has involved constructing the setup
(Figure 5) described above, interfacing it with the computer, and learning how to operate
it. One of the main challenges in tuning it was correctly aligning the beam with the EOM
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Figure 4: Internal microscope mirror setup. Shown here are the sample stage (1), 60times
water objective (2), dichroic mirror (3), laser input (4), fluorescent light source (5), and
imaging camera (6). This setup allows a single objective to be used as both an imaging
focus as well as a condenser for the laser light.
and adjusting the mirrors to direct the beam into the microscope objective centered and
normal to the objective face. This still poses issues for future use because it is a process
which takes time but specialized equipment is being built to make the process of alignment
easy for any user. Next, I tuned the EOM electronics to find the operating parameter at
which it operates as a fast shutter. This tuning must quickly be performed for each use of
the system for ablation
I have found that when at the quiescent operating point, the 3.8 W average power
input to the EOM is cut to ∼15 mW. However, it would be more desirable to have this
lower, cutting the average power to the region of µW so that the worm will be exposed
to minimal average power beyond the power from the pulses. Using a blank sample for
testing, I optimized the spot quality by adjustment of the mirrors and microscope focus.
Examples of the spot appearance are shown in Figure 6.
After optimizing the spot visually, I began to run preliminary tests to attempt ablation
using worms that have been genetically modified such that the AFD neuron in the pharynx
region of the body glows when exposed to fluorescent light. This fluorescent tagging is what
allows the laser to be aimed so as to minimize collateral damage.
For ablation, the worms are prepared by placing them on a pad of very viscous (10%)
agarose pad on a slide. A drop of either 10 µM levamisole solution or 10µm polystyrene
bead solution is introduced to immobilize the worm. These are both meant to immobilize
the worm. Levamisole is a chemical that functions as an sedative when the worm absorbs it.
The beads immobilize the worm by causing it to become jammed in a semirigid structure
that is created by friction and jamming. Thus far, I have found the levamisole to work
slightly better but the present method of slide preparation needs to be improved overall
because the 10% agarose is difficult to work with and the optimal levamisole concentration
for sedation of the worms is still unknown. A cover slip is then used to close the sample so
the worm does not dry out and act as the interface between the microscope objective and
the sample.
The sample is positioned on the microscope stage using a stepper motor such that the
neuron of interest is located where the laser spot will be when the shutter is opened. The
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Figure 5: Completed experimental setup used for ablation. Shown here are the laser (1),
EOM (2), directive optics and shutter (3), and the microscope and sample stage (4).
Figure 6: Shown here are images taken of the laser sport focused onto a blank slide of
water. The spot size is approximately 3µm in diameter. The exposure times are 200ms
(left) and 10ms (right). Note the good quality of the more intense spot on the right. This
is the region in which nonlinear absorption and damage to biological material occurs.
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frequency of laser pulses must be tuned down using the EOM so as to not cause heating or
other undesirable effects in the worm. Previous labs have found that frequencies of ∼1kHz
proves sufficient for ablation [2]. However, in the preliminary results, I have used repetition
rates of ∼80kHz to ensure successful ablation. The shutter is opened for a short period of
time, ∼1-2 s, to cause the damage and then closed. Again, I used a much longer exposure
times for the same reason. The results of one demonstration are shown in Figure 7.
Figure 7: Demonstration of ablation of AFD neuron in C. elegans. Shown here are fluorescent images taken before (left), during (center), and after (right) ablation. The images
are taken over the course of 3 seconds and the size of the neuron is ∼2 µm
.
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Spring Quarter Goals
From the previous results, it is evident that the system is functioning as expected. However,
further tuning can be performed and the operating procedure can be made more user
friendly. To this end, my immediate goals for the spring quarter are as follows.
• Measure the power attenuation at each stage of the beamline
• Work to minimize the quiescent operating point minimum average power
• Test the effect of GVD precompensator adjustments without autocorrelator
• Perform ablation using more realistic power settings (f ∼ 1 kHz, texposure ∼ 2 s)
• Perform ablation at different stages of worm development
• Test different variations of worm immobilization using levamisole
• Create documentation fortuning and use of system by future people using this setup
In addition to optimizing the working procedure, now that the system is in a functional
state, we have decided to perform a preliminary study using ablation of the AFD neuron
that I am presently using for testing of the system. This neuron has been studied by
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previous groups and is believed to be important for the worm’s sense of temperature [4].
The memory of a worm integrates over the course of its life and so it prefers to live
in a region of temperature in which it was born. Thus, when placed on a temperature
gradient, worms will in general sense there way back to a location in the gradient in which
the temperature is the same at which it was raised. The study will consist of using the
laser to ablate the AFD neuron in 10-20 worms and then comparing their movement on a
temperature gradient to that of the same number of control worms with no ablation.
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Progress Comments
I am satisfied with my current progress and feel that my project is being carried out
according to my initial plan. I feel that during this quarter, I will have sufficient time
to complete the initial intent of my project, that being to successfully and consistently
reproduce ablation, and create a setup that is user friendly for the lab. The difficulties I
am presently encountering are of the type I expected to have during this project and with
work. Further, I am pleased that there is the opportunity to pursue using this system to
perform a study and obtain results beyond experimental setup.
References
[1] Samuel Chung. Surgical applications of femtosecond lasers. Journal of Biophotonics,
2009.
[2] Samuel Chung and Eric Mazur. Femtosecond laser ablation of neurons in C. elegans
for behavioral studies. Applied Physics A, 2009.
[3] Joseph Steinmeyer et. al. Construction of a femtosecond laser microsurgery system.
Nature Protocols, 2010.
[4] Samuel Chung et. al. The role of the AFD neuron in C. elegans thermotaxis analyzed
using femtosecond laser ablation. BMC Neuroscience, 2006.
[5] Coherent Lasers. Operator’s manual chameleon ultra and chameleon vision diodepumped laser, 2008.
[6] A. Vogel. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Applied
Physics B, 2005.
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