Electrically Evoked Motions of the Isolated Mouse
Tectorial Membrane
by
Roozbeh Ghaffari
Submitted to the Department of Electrical Engineering and Computer
Science
in partial fulfillment of the requirements for the degree of
Master of Engineering in Electrical Engineering and Computer Science
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2003
@
Massachusetts Institute of Technology 2003. All rights reserved.
Author......
Department of Electrical Engineering and Computer Science
September, 2003
Certified by...
. . . . . . . .
.
.
.
..
. .- .
* ....
... V
-. .
- -
. ............
Dennis M. Freeman
Associate Professor
Thesis Supervisor
..-.. .. ... . . .. . .. .
Arthur C. Smith
Chairman, Department Committee on Graduate Students
A ccepted by (
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Electrically Evoked Motions of the Isolated Mouse Tectorial
Membrane
by
Roozbeh Ghaffari
Submitted to the Department of Electrical Engineering and Computer Science
on September, 2003, in partial fulfillment of the
requirements for the degree of
Master of Engineering in Electrical Engineering and Computer Science
Abstract
We discovered motion during application of AC voltage (0.8 V peak amplitude, f=1
kHz) on the surface of the isolated mouse tectorial membrane (TM). The TM's motion
response, which contained an average peak amplitude of 4 nm (in 5 TM preparations)
was measured using a novel atomic force sensing (AFS) technique (Rousso et al, 1997).
A 2-D lateral mapping of motion at several points on the TM surface shows that the
TM expands near the negative electrode and contracts near the positive electrode
with a stationary pivot point between the two electrodes.
Lowering the pH in the bath surrounding the TM from 7.3 to 4.07 decreased
the maximum amplitude of displacement from 4 nm to approximately 2.5 nm while
lowering the bath pH from 4.07 to 3.96 caused the TM to undergo a 7r phase shift in
its motion response. Based on this data, the TM has an isoelectric point and pKa
near pH 4.01. This supports the model that the TM motion response is altered by
the state of ionization of charge groups in the TM, which varies with bath pH.
Thesis Supervisor: Dennis M. Freeman
Title: Associate Professor
3
4
Acknowledgments
There are many people who have been a source of immense help and support to help
me reach this point in my journey. First and foremost, I would like to thank my
research advisor, Denny Freeman. I vividly remember walking into his office two
years ago when he took time out of his busy schedule to explain to me his research
in hearing. He not only explained his research but he also went in depth about the
different projects that he felt I could tackle. As an undergrad, I knew very little about
hearing research, and I was even more clueless about running successful experiments;
and so, this was a tremendous confidence boost for me to have Denny support me in
this way.
The past 2 years in Denny's lab have helped me realize how much there is to learn
in order to have a solid physical understanding of the world. This understanding
transcends beyond hearing research and is more fundamentally about the way I think
and perceive the world. It has helped me question things that two years ago, I would
have just accepted. It is comforting and at the same time exciting to know that this
is only the beginning of the journey. My goal is that one day I may be able to tackle
problems the way Denny does.
My lab co-workers have also been instrumental in the completion of my Master's
Thesis:
Kinu (aka. Kobe, Tokyo Ninja) for always being there for me as a mentor (who
singlehandedly convinced me to apply to my PhD program after putting up with my
stubbornness) and as a great friend. Without her support and friendship, I would
not be pursuing my PhD today.
AJ (Shaq Diesel), for being a big brother to me, and also for taking the time and
patience to answer all my questions.
Salil (aka. Brownsides) for also being a big brother, a mentor (with everything
ranging from Starbucks, to travel in Europe, to MEMS research), and a great friend.
Luke (aka. Lukovich) for teaching me how to microfab and always making me
question the seemingly obvious.
5
Amy for helping me get started with the TM and research.
And the rest of my lab crew (Andy, Stan, J Ryu, and honorary member, Laila).
My undergraduate advisor, Dr. Roger Mark has also offered helpful advice on
how to plan my future goals around my research endeavors.
This particular project would not have been possible without the help of Itay
Rousso, who has been a great friend and mentor. Itay opened an entirely unexpected
window of opportunity to my research, which has given me direction for my PhD
research. I hope to continue working with him for many years to come.
My roommates over the years [934: Emelio (aka. mookie), Jamy (aka. Haitian
sensation), Nik] and my MIT and Harvard friends [Ab, Ayanna, Machu, G, Jeloni,
Karl Reid, MITE2S '01, '02, '03] have been there for me to ease off the stresses and
demands of life at MIT.
Finally, my family's support has also been invaluable throughout these two years
and throughout my life. My mom and dad, who have dedicated their lives to making
sure I am happy and my lil' brother, Soran, who I miss every single day while I am
away from home, thanks for all the love.
And to the ONE: I hope to share this and everything else that may follow with
you. As I said, the past 2 years are just the beginning...
6
Contents
1
Introduction
11
1.1
Composition of the TM. . . . . . . . . . . . . . . . .
12
1.2
Observations of Connective Tissues . . . . . . . . . .
13
1.3
Previous Observations of the TM
. . . . . . . . . . .
14
1.3.1
Micropipet Method . . . . . . . . . . . . . . .
14
1.3.2
Two-Bath Method
. . . . . . . . . . . . . . .
14
1.3.3
Osmotic Response Caused by Changes in pH .
16
Specific Aims . . . . . . . . . . . . . . . . . . . . . .
17
1.4
19
2 Methods
Experiment Chamber . . . . . . . . . . . . . . . . . .
19
. . . . . . . . . .
19
2.2
Isolated TM preparation . . . . . . . . . . . . . . . .
22
2.3
Experiment Parameters . . . . . . . . . . . . . . . . .
22
2.4
Atomic Force Sensing Technique . . . . . . . . . . . .
23
2.5
pH Titration. . . . . . . . . . . . . . . . . . . . . . .
23
2.6
C ontrols . . . . . . . . . . . . . . . . . . . . . . . . .
25
2.1
2.1.1
3
Microfabrication Technique
27
Results
3.1
3.2
. . . . . . . . . . . . .
27
. . . . . . . . . . . . .
27
. .
. . . . . . . . . . . . .
28
Controls . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .
30
TM Motion in Response to Voltage
3.1.1
2-D Lateral Mapping of Motion
3.1.2
Effect of pH on TM Motion
7
4
5
Discussion
33
4.1
Seesaw Motion Along Longitudinal Length . . . . . . . . . . . . . . .
33
4.2
Surface Charge Model
. . . . . . . . . . . . . . . . . . . . . . . . . .
34
4.3
Radial and Longitudinal Motion of TM . . . . . . . . . . . . . . . . .
35
4.4
Implications of Motion Variations Caused by Changes in pH . . . . .
35
4.5
Implications of Volume Changes on Motion . . . . . . . . . . . . . . .
37
Conclusions
39
8
List of Figures
1-1
. .. .. .. ... ... ... ..... .. ... ... .. ... ... ..
12
1-2
. .. .. .. .. .... ... ..... .. ... ... .. ... .. ...
13
1-3
... .. .. .. ... ... ...... .. ... ... .. ... .. .. .
15
1-4
... .. .. .. .... ... ..... .. ... ... .. ... ... ..
17
1-5
... . .. ... ... ... ...... .. ... ... .. ... .. ...
18
2-1
20
2-2
21
2-3
24
3-1
28
3-2
29
3-3
30
3-4
31
4-1
34
4-2
36
9
10
Chapter 1
Introduction
The tectorial membrane (TM) sits in a critical position in the inner ear directly above
the sensory hair cells. It spans the entire length of the cochlea and is approximately
100 pm in width and 30 pm in thickness. Based on its position overlying the hair
bundles in the inner ear as illustrated in Figure 1-1, it is widely accepted that the TM
plays a key role in the micromechanical stimulation of hair cells (Freeman et al., 2003).
Recent findings in genetic research have further strengthened this reasoning. Genetic
mutations of proteins in the TM have been found to cause auditory impairments. For
instance, humans lacking the COL11A2 gene, which encodes a specific collagen in the
TM, have a 40-60 dB hearing loss (McGuirt et al., 1999) further suggesting that the
TM is important in hearing. However, studies investigating the TM are still in their
infancy due to experimental difficulties associated with the TM's transparency, small
size, and fragility.
The classical view states that the TM generates a shearing force on the hair cells
relative to the position of the basilar membrane (Johnstone and Johnstone, 1966).
However, recent findings about the TM's composition suggest that this gelatinous,
acellular, charged structure (Freeman et al., 2003) may play a more complicated role
in cochlear micromechanics.
11
longitudinal fibrils
T
radial fibrils
Figure 1-1: Schematic showing the tectorial membrane's location in the cochlea in
relation to the outer hair cells (OHC), inner hair cells (IHC), and the spiral limbus
(SL), which anchors the TM. The TM's skeletal structure is comprised of radial and
longitudinal fibrils that are visible in light microscopy.
1.1
Composition of the TM
An understanding of the TM's chemical composition and structure is critical to uncovering its role in hearing. The solid composition of the TM, which makes up 3%
of its total weight, is comprised of glycosaminoglycans (GAGs) and proteins. The
rest of the TM is water (97%). The solid constituents (GAGs and proteins) both
have ionizable fixed charge groups in the TM. This chemical makeup of the TM is
analogous to the structure of polyelectrolyte gels. Therefore, a polyelectrolyte gel
model has been applied to the TM (Weiss and Freeman, 1997). This gel model has
two parameters: the fixed charge concentration (cf) of the TM and its bulk modulus
(n). The cf is the amount of ionizable non-mobile charge groups contained within the
macromolecular structure per unit volume of material. Mobile ions and fixed charge
inside the TM are illustrated in Figure 1-2. The distributed compressibility of the
TM is characterized by r.. Like all gels, cf and r, can effect osmotic, mechanical, and
chemical forces inside the TM.
The goal of this project is to learn about the material properties (cf and r, relationship) of the TM using a novel atomic force sensing technique in an effort to
uncover the TM's role in hearing.
12
Fixed ionizable groups
ID positive
B negative
Oneutral
0
6 Gel
Mobile solutes
~ cations
eanions
ouncharged
Figure 1-2: The structure of the TM is modeled as a polyelectrolyte gel immersed in
a surrounding bath. The TM has predominantly negatively charged ions fixed along
the collagen matrix with mobile ions free floating inside the gel (Freeman et al., 2003).
1.2
Observations of Connective Tissues
Methods to measure material properties in several connective tissues like the TM
have been successful. In the case of cartilage, cylindrical specimens with dimensions
on the order of 1 cm in diameter and 1 mm in depth are typically placed in Ussing
chambers for electrical transport studies.
The material properties of cartilage have been extensively studied. Eisenberg
and Grodzinsky have shown that the application of a compressive force generates a
streaming potential across cartilage (Eisenberg and Grodzinsky, 1985). In connective tissues like cartilage and TM, the negative fixed charge groups and interstitial
fluid that contains an excess of positive counterions, balance each other to maintain
electroneutrality. In streaming potential experiments, Ag/AgCl reference electrode
compresses the cartilage tissue against a porous Ag/AgCl electrode, which in turn
forces fluid through the porous electrode. This fluid motion creates a drag on the
mobile ions and an overall charge separation between the fixed charge groups and the
mobile counterions, which is defined as a streaming potential.
The reverse procedure is to drive the tissue with voltage instead of using a mechanical probe. Grodzinsky and Sachs developed a three-dimensional model to predict the
motion response of a finite-thickness layer of cartilage to a sinusoidal current (Sachs
and Grodzinsky, 1989). And so, cartilage is a well-studied connective tissue that has
been shown to respond to electrical stimulus. This finding that connective tissues can
13
respond with motion to electrical stimulus is applied to the TM in this thesis.
1.3
Previous Observations of the TM
Investigations attempting to quantify the charge groups on the GAG molecules have
been a focus of numerous TM research projects. However, typical electrical measurement tools like the Ussing chamber are incompatible with the TM because of the
TM's small size and fragility during mechanical manipulation. The main overarching challenge is to engineer an experimental chamber and technique that provides a
stable environment for the TM by minimizing exposure to air, fluid flow, and mechanical manipulation. The chamber schematics in Figure 1-3 illustrate the previous
experiments designed to measure cf in the TM.
1.3.1
Micropipet Method
The micropipet method was the first set of electrical experiments that yielded potential measurements of the TM. However, these potentials were not stable nor repeatable. The reason for the instability was attributed to the pipet causing leaks between
the reference solution in the bath and the test solution in the pipet. When the pipet
tip is inserted into the membrane it separates the fibers of the TM and this may allow
solution to flow from the bath to the pipet. This results in the whole system shorting out. The potential recordings were further complicated by junction potentials at
the boundary between the pipet and the TM (Adrian, 1956). The advantage of this
apparatus is that the TM remains submerged in solution.
1.3.2
Two-Bath Method
The two-bath technique takes the approach taken by researchers who study cartilage. The main advantage of this technique over the micropipet method was in its
measurement stability. By exposing the TM to two separated solutions that differed
in ionic strength, two distinct junction potentials were established. The difference
14
-
pipet+?
VVbath +
Figure 1-3: Schematic diagram of the micropipet method (top) and two-bath method
(bottom). The illustration of the micropipet method depicts a pipet piercing the
surface of the TM. The diameter of the micropipet at the tip is between 1-10 /im.
The potential between the micropipet and the electrode in the bath is Vpjpet = VIR VIT
+ VB. VIT and VIR are the interfacial potentials between the TM and reference
solutions and VB is the potential through the bulk of the TM. An elevation view of
the two-bath technique (bottom) is also shown with the TM draped across. Ag/AgCl
electrodes are positioned in the two baths and fluid streams down the two baths. The
only link between the two baths is the TM. The potential between the two baths is
Vbath = VIR - VIT + VB (Freeman et al., 2003).
15
between the junction potentials in the two baths yielded the potential across the
TM. However, the measured values of cf using the two-bath method were an order of
magnitude greater than the predicted estimates (Weiss and Freeman, 1997). Several
factors could have been responsible for this discrepancy.
The TM was exposed to air in the two-bath method. Air exposure has been shown
to cause potential fluctuations in gels (Englehart, 2002) like the TM. The potential
difference increased with time. The fluctuations in potential measurements were also
coupled with shrinking in the volume that resulted from air exposure.
Furthermore, variations in ionic concentration has the effect of influencing several
properties of the TM. Based on the gel model, the cf is dependent on ionic concentrations, conformational changes, and on osmotic pressure changes. None of these
variables are mutually exclusive, which means that variations in ionic concentrations
lead to several fundamental changes inside the TM according to Freeman and Weiss
(1997) polyelectrolyte gel model.
1.3.3
Osmotic Response Caused by Changes in pH
The material properties of the TM were also measured using an indirect technique.
The pH experiment by Freeman et al. (1997) avoided air exposure problems by tracking the motion of beads on the TM's surface using a computer-controlled automated
system (Shah et al., 1995). Changes in pH cause changes in the size and shape of the
TM. Freeman et al. (1997) showed that the TM thickness swelled in highly acidic and
basic solutions. In mildly acidic solutions, the thickness decreased by approximately
1% (Freeman et al., 1997). The gel model predictions shown in Figure 1-4 are plotted
with the experimental results. The gel model parameters (Cf and rK) are described
in Figure 1-5, which shows how the sign of the fixed charge varies with pH. According
to the gel model, the isoelectric point of the TM is around pH 6, which coincides with
the point of lowest thicknessin the pH experiment. Although the model prediction for
the isoelectric point matches the value found experimentally, there are several variables that may effect the location of the isolectric point that the model did not take
into account. For instance, Freeman et al., (1997) saw not only changes in thickness
16
80-
Gel model
S60-
Measurements
2400
-200
5
7
pH
9
Figure 1-4: Comparison of median percentage change in thickness predicted by the
gel model of the TM as a function of pH plotted with theexperimental data measured
by Freeman et al. (1997) (Freeman et al., 2003).
but also rapid fluctuations in the radial dimension. Multiple changes in the volume
parameters could influence the location of the isoelectric point.
1.4
Specific Aims
In this thesis, a new technique at the interface of MEMS (microelectromechanical
systems) research and atomic force sensing (Rousso et al., 1997) probes the material
properties of the TM by measuring induced motion. This technique has the advantage of probing the TM's cf and rK gel model paramaters directly in contrast to the
pH swelling experiment. Furthermore, this technique has the potential to directly
measure the pKa of the TM without having to infer it from changes in the volume.
The big picture goals of this project are to:
A) Develop a method to apply an electrical stimulus to the TM and measure the
17
11
carboxyl groups
C
charged negatively
0
5
inn collagen
chared
psitielycharged
0 0-1
c> E-1
"0E
'P_--
-
(Z
a>
iL
-o 20
9
11
negatively
carboxyl and
sulfate groups
in GAGs
charged negatively\
-
amino groups
in collagen
charged positively
Figure 1-5: The dependence of fixed charge on pH according to a model based on
the composition of the TM. The magnitude of the fixed charge concentration increases at high and low pH. The gel model predicts that the magnitude of the volume
should increase as the magnitude of the fixed charge is increased, which was found
experimentally by Freeman et al. (1997).
resulting motion.
B) Characterize how motion depends on parameters of the electrical stimulation
(frequency and amplitude) and pH.
C) Develop a mathematical model that relates electrical response properties to
the underlying molecular properties (such as cf and r).
D) Compare the model to the electrical properties that exist in the live cochlea
to see if the motion measurements have physiological relevance.
My thesis addresses points A and B. The next task following this thesis is to
develop a robust model for the induced motion in an effort to discover if the motion
is relevant in hearing.
18
Chapter 2
Methods
2.1
Experiment Chamber
The experiment chamber was comprised of the TM draped across gold microfabricated
electrodes that supply voltage as shown in Figure 2-1. The electrodes were microfabricated on glass slides using gold "lift-off" microfabrication technique (Whitesides
et al., 2001). The glass slides were used because they allowed visual analysis of the
TM during atomic force sensing (AFS) motion measurements, which enabled precise
positioning of the atomic force microscope (AFM) tip on the TM's surface.
2.1.1
Microfabrication Technique
A number of "lift-off" methods exist for different biological and microelectronic applications. For the purposes of this experiment, a multilayer technique consisting of
titanium and gold is applied to a glass substrate. A layer of titanium metal with a
thickness of 0.025 pm acts as the adhesion layer on the surface of the glass. Gold
metal, with a thickness of 0.125 pum is then deposited on top of this thin adhesion
layer. The steps taken to create microfabricated electrodes are illustrated in Figure 22.
The detailed steps taken to create the Au-Ti pattern on glass are as follows:
1. Hard bake clean glass slide for 10 minutes in 130 degrees Celsius.
19
glass slide
_TM
Gold Electrodes
100 Jim
Gold E ectrodes
Figure 2-1: (Left) Schematic drawing of the TM (not to scale) attached to the glass
floor of the e-field chamber, which contains parallel gold-electrode patterns on the
surface of glass. (Right) A light microscope image at 1oX magnification of the TM
attached to the e-field chamber. The leads of the gold electrodes are separated by
125 pm in the region where the TM is draped. The TM and the tips of the electrodes
were immersed in AE solution.
2. Coat glass slide with AZ 4620 Photoresist with a spin time of 40 seconds at a
spin rate of 2500 rpm.
3. Soft bake the glass slide for 45 minutes in 90 degrees Celsius.
4. Place microelectrode pattern on glass slide and expose to UV radiation for 45
seconds.
5. Rinse the glass slide in AZ 440 Developer solution for 5 minutes.
6. Rinse in deionized water to remove excess resist from patterned regions.
7. Soft bake for 30 minutes in 90 degrees celsius.
8. Expose the glass slide to oxygen plasma for 2 minutes for further cleansing of
patterned area.
9. Place glass slide in E-beam machine and expose it to titanium and gold metal
vapors (detailed description of this process can be found in the Exploratory Materials
Laboratory (EML)).
10. Place glass slide in acetone solution and expose to ultrasonic waves transmitted
through deionized water to promote the "lift-off" of photoresist and Au-Ti sitting on
top of the photoresist.
20
Glass
1. Prebake glass (130 degrees Celsius)
2. Apply AZ 4620 photoresist
3. Bake resist on glass (90 degrees Celsius)
- Photores'ist
-
-
-
-
Glass
1. Place electrode mask on top of slide
2. Expose to ultraviolet radiation
3. Immerse in AZ 440 developer solution
I Po0tarsi
Glass
' I,
/
1. Place chamber in E-Beam
2. Expose to titanium (Ti) metal for the adhesion layer
3. Expose to gold (Au)
q-foifrtisti:
I
Glass
1. Place chamber in acetone solution
2. Expose chamber to ultrasonic waves
transmitted through deionized water
Au-Ti
0.125jpm
Glass
t
Figure 2-2: A schematic outline of gold lift-off patterning. The process begins with a
clean glass slide. A 1 pm layer of photoresist is evenly spread across the surface of the
glass slide using a spinner. The photoresist is baked and then exposed to ultraviolet
light through a mask. The exposed regions of photoresist are then removed using
developer solution. This leaves the patterned regions free of photoresist. The next
step is to place the chamber in the e-beam machine to deposit Au-Ti. Once the
Au-Ti deposition is complete, the chamber is washed in acetone solution to remove
the excess Au-Ti sitting on top of the photoresist.
21
Overall, the entire "liftoff" process takes approximately 8 hours to complete with
about a 75% success rate. During a single fabrication trial, several of these chambers
can be fabricated depending on the availability of the equipment in the EML facility.
2.2
Isolated TM preparation
TMs were isolated from adult male mice (Shah et al., 1995) after asphyxiation by C0 2 .
The cochlea was dissected and removed from the temporal bone using a technique
similar to that reported by Abnet and Freeman (2000). The cochlea was then placed
in artificial endolymph (AE: 174 mmol/L KCl + 0.02 mmol/L CaCl 2 + 5 mmol/L
HEPES + 2 mmol NaCl at pH 7.3). A scalpel blade was used to expose the organ of
Corti. Using dark field illumination under a dissecting microscope, the TM became
visible and was isolated with an eyelash manipulator. We studied the basal sections
of TM in these experiments.
Once isolated from the cochlea, the TM was transferred to the tips of the gold
electrodes on the experiment chamber using a micropipet filled with AE solution. The
TM is not prone to stick to glass and gold surfaces without adhesive coating. However,
Cell-Tak (BD Biosciences, Bedford, MA.) makes the TM stick and remain stable
during sensitive motion measurements. The next step was to quickly position the
TM on the electrodes using Cell-Tak. An eyelash manipulator was used to manuever
the TM and gently place it in position over the electrodes. Adhesion of the TM to
the surface of the Cell-Tak occurs instantly.
2.3
Experiment Parameters
AC voltage with a peak amplitude of 0.8 V with a frequency of 1 kHz was applied
to the TM in the AE fluid in the experiment chamber. The voltage supplied by the
two electrodes differed in phase by 180 degrees. The resistance (Rt0 ) between the
two electrodes with AE fluid was measured to be 300 ohms during application of AC
voltage. Motion was measured using the AFS technique once voltage was applied
22
through the chamber.
2.4
Atomic Force Sensing Technique
The AFS (Rousso et al., 1997) technique was used to measure electrically induced
motion of the TM. This technique enabled very precise direct motion measurements
with sub-angstrom resolution.
In our experiment, the AFM tip was engaged on the surface of the TM in contact mode. The nature of these of AFM probes is such that any motion in the tip
is reflected in the cantilever position. The model DNP AFM probe from Digital
Instruments (Santa Barbara. CA.) was chosen for our measurements. The probe's
cantilever is made of silicon nitride, is triangular and approximately 200 pm in length.
The tip chosen for our experiments was very sensitive and flexible with an ultra-low
spring constant of 0.06 N/m.
Once the TM was excited, the motion of the TM caused the tip on the surface to
bend with the TM motion. Figure 2-3 is a schematic of the AFM apparatus, which
shows how the bending of the cantilever caused signals to be sent to the position
sensitive detector (PSD). During each experiment, the entire AFM cantilever was
viewed from below the chamber with an inverted light microscope with 10X resolution.
The ability to make motion measurements at a single point on the surface of the
TM allowed for 2-D lateral mapping of motion. In the lateral mapping experiment,
the AFM tip was engaged on several points on the TM's surface between the two
electrodes. Motion was measured at each point of engagement to generate a 2-D view
of the surface motion.
2.5
pH Titration
TM motion was measured as a function of pH. Test solutions with pH ranging from
3.62 to 7.3 were prepared by adding KOH or HCl until the pH reached the intended
value. All solutions were prepared with the same ionic strength. The TM was in23
Diode Laser
(A)
AE Solution-.
(B
AFM Cantilever
Position Sensitive Detector
Diode Laser
Position Sensitive Detector
AFM Cantilever
AE Solution
TM
E-Field Chamber-s.
E-Field Chamber
Oscillo
Oscilloscope
V
OFF
cope
V=ON
f = 1 kHz
Figure 2-3: Schematic of the AFM experimental apparatus. (A) TM is at rest when
no voltage is applied. The diode laser reflects from the cantilever to a position on
the position sensitive detector, which is calibrated as the zero displacement point.
(B) The AC voltage (0.8 V peak amplitude) causes the TM to expand and contract
at the negative and positive electrodes, respectively. This behavior is recorded using
the AFS technique as the AFM tip bends in response to changes in surface height.
This bending causes the diode laser to reflect off of different points on the position
sensitive detector, which correspond to the precise vertical displacement of the TM
during voltage application.
24
cubated in each solution for 10 minutes prior to AFS measurements. Motion was
recorded at each pH. The data were then curve-fitted using the Henderson-Hasselbach
equation:
AZ
1+
AZmax
10n(pKa -pH) '
(2.1)
where AZ is the displacement plotted on the vertical axis in a AZ versus pH
titration plot, AZmax is the maximum displacement, n is the number of protons
participating in the transition, and the pKa is the midpoint of the titration. During
each titration experiment, the pH of the bath was returned to 7.3 at the conclusion
to measure reversibility.
2.6
Controls
The sub-angstrom resolution of the AFS technique applied to the TM in fluid introduces several sources of artifacts that may potentially cause unwanted motion that
may be misrepresented as TM motion. We applied several control experiments prior
to each TM measurement to ensure the TM motion was real.
1) Artifacts due to refraction: The TM is immersed in AE solution. The diode
laser reflects off the AFM cantilever through this liquid medium. The fluid could
potentially cause the the laser to reflect differently due to refraction, which in turn
will alter the measured motion of the TM. We engaged the tip away from the TM
and measured zero motion signal while the voltage was on to ensure fluid motion was
not effecting the measured motion. Since there was zero motion signal when the tip
was away from the TM, this revealed that the motion of fluid was not contributing
significant artifacts to our measurements.
2) Charge on AFM tip: The interaction of charges in the AE solution with the
AFM tip may be another source of noise. There is the possibility that ionizable charge
groups may attach to the AFM tip and may cause repulsion and attraction effects that
may be misrepresented as motion. We positioned the tip a few micrometers above
the surface of the TM and recorded zero motion signal before each AFS experiment
25
to ensure that this was not occuring.
3) Motion of the electrodes: We engaged the tip on the surface of the electrodes
to ensure that electrodes were stationary and not causing mechanically as opposed to
electrically induced motion of the TM. The electrodes wear out over time. This test
was critical to ensure that the electrodes were functioning properly.
26
Chapter 3
Results
Motion responses of the isolated TM were discovered using the AFS technique during
application of AC voltage. We describe results obtained from 3 TM preparations
isolated from different mice.
3.1
TM Motion in Response to Voltage
Results obtained from a single point on the surface of the TM in close proximity
to one of the electrodes show that there was vertical contraction and expansion (on
the order of 2 to 5 nm) in response to 0.8 V of AC voltage. More specifically, the
TM expanded during application of negative voltage and contracted during positive
voltage. Figure 3-1 shows that a TM sample had a peak expansion of 5 nm and a
ir phase shift relative to the frequency of the AC driving voltage. The 5 nm peak
in the motion response occurred approximately at -0.8 V whereas the maximum
contraction (defined as negative displacement) was at +0.8 V.
3.1.1
2-D Lateral Mapping of Motion
The AFM tip was engaged at 5 points along the longitudinal dimension of the TM
between the two electrodes. Measurements of motion were recorded at each of these
points shown in Figure 3-2. The points where the tip was engaged were chosen to
27
-0.8
4-
Figure 3-1: (Solid)
TM motion in response to voltage as
a function of time.
The input
2-
T
0-
-(Dotted)
0.0
-0.4
-0.8
-6-
0
1
2
3
45
5the
driving
voltage
is
also plotted to show
that the motion signal recorded using
the AFS technique
is out of phase with
driving voltage.
Time [msec]
be parallel to the contour of the Hensen's stripe. The motion response was greatest
in amplitude at the two points closest to the two electrodes (a and e). These two
points were also approximately 7r out of phase with each other. The motion response
dropped at the interior points between points a and e to the extent where at point c,
the motion response was close to zero.
3.1.2
Effect of pH on TM Motion
We next measured variations in the motion response of the TM at a single point due
to changes in pH. Lowering the pH systematically from 7.3 to 4.07 caused a significant
reduction of the motion amplitude. Figure 3-3 shows the peak amplitude of the motion
response of the TM as a function of pH. The amplitude of the motion remained stable
up to pH 4.5, at which point, we began to see it decrease significantly. The motion
response at pH 4.07 was approximately half the size of the motion measured at pH
7.3.
Once the pH of the bath was lowered below 4, the AZ became negative as illustrated in Figure 3-3. This signified a phase shift in the motion response relative
to the motion measured at pH 7.3. The phase shift was approximately 7r. The pKa
28
0-2
2-b
0-
2-C
E
-2-2-
0-
2-d
0
-22-
260 gm
Gold Electrodes
-2.
0
1
2
3
4
5
Time [msec]
Figure 3-2: 2-D lateral mapping of TM motion as a function of time. (Left) The points
marked on the TM (a through e) are the positions where the AFM tip was engaged
and motion was measured during voltage application. (Right) The motion plots (a
through e) correspond to the motion at each point marked on the TM photograph.
At points a and e, the motion responses reached maximum peak amplitudes. The
motion dropped in amplitude as the tip was moved close to the midpoint between
the two electrodes. There was a consistent 7r phase difference between the motion at
point a and point e.
29
54-
(
3-
2
~(4.O7, 2.8 nm)
+ (recovery)
21-
-2-
(3.96, -1.9 n m)
-3
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
pH
Figure 3-3: Changes in motion response of the TM during perfusions of acidic solutions. The magnitude of TM motion decreased at low pH values. Below pH 4.07, we
recorded a ir phase shift in the TM's motion at which point the TM flipped its behavior at the negative and positive electrodes. The pKa of the TM was approximately
4.01. The red point shows the peak amplitude when the pH was returned to 7.3 at
the conclusion of the experiment.
for this titration was approximately 4.01 using the Henderson-Hasselbach equation
applied to the 6 data points shown in Figure 3-3. This experiment was repeatable
with 3 separate samples of TM, with the pKa value consistently being between pH
3.97 and 4.07.
Once the pH was returned to 7.3 at the conclusion of the titration, the motion
response of the TM recovered in phase; but the amplitude decreased by approximately
50% compared to the initial motion measured at the beginning of the experiment.
The peak amplitude measured at pH 7.3 initially was 4.2 nm. After the titration, the
peak amplitude at pH 7.3 was approximately 2 nm.
3.2
Controls
The motion response of the AFM tip was measured when it was engaged a few
micrometers above the TM surface. The motion was approximately 2 orders of mag30
I
I
I
I
I
I
I
I
1
I
0.05E
Ca
0
03
0.
0.05
0
I
I
I
I
I
I
I
I
I
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1
time (msec)
Figure 3-4: Motion response of the AFM tip engaged in AE solution not in contact
with the surface of the TM (blue) plotted with respect to the input driving voltage
(red). The motion response is on the order of 0.05 nm in peak amplitude. This is
100 times smaller than the motion measured on the surface of the TM (between 2-5
nm).
nitude smaller than the motion measured on the TM surface. Figure 3-4 shows that
the motion does not have the phase qualities and is small and haphazard. This was
defined as the noise floor in our experiments.
31
32
Chapter 4
Discussion
It has been demonstrated that the TM expands and contracts in response to AC voltage stimulus. The TM motion follows the polyelectrolyte gel model, which predicts
that it contains net negative fixed charge. For example, over the negative electrode,
the negative fixed charge repels and as a result causes the TM to expand, while at
the positive electrode, the fixed charge causes the TM structure to contract closer to
the electrode.
4.1
Seesaw Motion Along Longitudinal Length
Further analysis of the TM's motion response at several points along its length revealed that it displays a seesaw motion. The center point between the two electrodes
is a node with minimal displacement while the motion near the electrodes is out of
phase and of maximum displacement, thus giving it a seesaw effect. This motion is
due to the vertical component of the electric field, which has a maximum amplitude
at the electrodes. The vertical component of the electric field approaches zero at the
midpoint between the electrodes. The midpoint coincides in position to the location
of the node in the motion response. Based on this analysis, the vertical component
of the electric field may be causing the motion of the TM. Hence the 2-D mapping
of motion showed that sections of the TM sitting on the electrodes exhibit maximum
displacement as a result of maximum electric field presence in the vertical direction
33
AE=
---
+4 nm
t=0
..._t
---
Z
l_
- nm
TM
glass
1V
VL
time (msec)
1V
VR
1V
\1V
time (msec)
Figure 4-1: Schematic of TM induced motion. The two electrodes (yellow) apply
sinusoidal voltage that are 180 degrees out of phase with each other. The measured
motion at different time intervals during this voltage application is like a seesaw phenomenon, in which the TM lying over the side with the +0.8 V application contracts
while the opposite side at -0.8 V expands. The t=0 point in time is defined with
zero voltage and hence, no induced motion across the TM. The TM at this point in
time is in equilibrium. VR and VL represent the input voltages at the right and left
electrodes, with the particular instances in time marked red, black, and blue.
while the node point occurs where the vertical component of the electric field is close
to zero as illustrated in Figure 4-1.
4.2
Surface Charge Model
The induced motion may be explained by examining the surface charges on the TM.
For the surface charge model, there has to be a potential difference between the inside
of the TM and the surrounding bath. This difference in potential is represented as
a step function, as there is a dramatic increase in voltage in the bath relative to the
TM. Analysis of the potential difference, electric field, and charge at a single point
on the surface of the TM reveals that there is a build up of net negative charge
inside the TM compared to a net positive charge on the outside surface of the TM.
This characterization is illustrated in Figure 4-2, which shows how the electric field
and charge distribution at a single point on the TM surface is derived from the
potential step function. This surface charge model can be extended to include the
34
entire surface area of the TM. However, it is unlikely that this surface charge can
explain motion on order of a few nanometers since the debye length in solutions
similar to the composition of the TM is on the order of 4 nm (Weiss, 1996).
4.3
Radial and Longitudinal Motion of TM
We have thus far examined the motion of the TM in the vertical direction, which leaves
the question: how does the TM behave in the radial and longitudinal directions during
voltage application. It is likely that the TM is moving in multiple dimensions based on
the electric field existing multiple directions in the vertical and longitudinal directions.
The motion measured in this experiment, which is only in the vertical direction, may
be a component of a more complex motion exhibited in various directions by the TM.
4.4
Implications of Motion Variations Caused by
Changes in pH
Changes in bath pH cause changes in the material properties of the TM according to
a 1997 study (Freeman et al., 1997), which examined the osmotic response of the TM
to changes in pH. Hence it is likely that the pH-induced motion changes of the TM
are a result of changes in the fixed charge concentration of the TM. Depending on the
bath pH, the GAGs and collagen macromolecules carry the fixed charge concentration
of the TM. The GAGs in the TM contain keratine sulfate and chondroitin sulfate,
which contribute to the negative fixed charge above pH 7.3 (Freeman et al., 1997). The
collagen matrix, on the other hand, contains amino groups that provide the matrix
with positive ionizable charge (Freeman et al., 1997) at acidic pH levels. Lowering the
pH causes neutralization of the negative fixed charge groups and causes the amino
acids to dominate (Freeman et al., 1997). Therefore, at pH 3.96 and lower, there is a
7r phase shift in the motion response because the net fixed charge positive.
The steady drop in amplitude of the TM motion response between pH 7.3 and
4.07, suggests that the negative fixed charge groups were steadily neutralized. The
35
A
boundary
a
bath
x
TM
E=
b
x
VEA
VE
=-v
2
@= p/E
C
I'x
Ir
Figure 4-2: (a) There is a potential difference between the TM and the surrounding
bath, which is approximated as an abrupt change in voltage. (b) The electric field
(E) is derived from the potential difference using the fact that the E is the negative
gradient of the potential (D). (c) The charge is found by taking the gradient of E. At
a single point, there is a doublet, which is depicted as two impulses one on each side
of the boundary. The impulses demonstrate that there is negative charge in the TM
and positive charge in the bath.
36
change in phase that occurred at 3.96 indicates that there is an isoelectric point
between pH 4.07 and 3.96. At this point, the fixed charge concentration is zero and
there should be no motion exhibited by the TM. This value is close to the pH 4.3
value reported in a study by Kronester-Frei (Kronester-Frei, 1979), which utilized a
different technique that examined the relation between TM thickness and pH.
This value does not agree with the pKa in the pH swelling experiment (Freeman
et al., 1997). In comparing the two experiments, it is important to note that the AFS
technique is a more direct measure of the the TM charge properties. The 7r shift in
phase can be explained by a sign change in the TM fixed charge, whereas it is much
harder to deduce changes in sign from a volumetric standpoint since several structural
parameters, in addition to the thickness, vary. Although this experiment is a more
direct measure of the charge properties of the TM, it is important to repeat the pH
experiment with the two-bath setup to help qualify the values measured using the
AFS technique.
At the conclusion of the pH titration experiments, the pH was raised back to
pH 7.3 to check for reversibility. However, the TM peak amplitude was reduced by
approximately 50% at pH 7.3 post titration compared to the value obtained before
the pH was lowered.
One possibility for the source of this irreversibility is that
lowering pH has irreversible mechanical effects on the structure of the TM or it may
be an irreversible effect on the chemical makeup (Freeman et al., 1997), in which case
proteins are irreversibly denatured as a result of low pH.
4.5
Implications of Volume Changes on Motion
It is important to note that varying the pH of the bath can induce TM volume
changes and variations in the TM volume can impact the motion. More specifically,
when the TM swells, it may display smaller motion behavior as the fixed charge
concentration drops, whereas it may display higher motion when it shrinks and the
fixed charge concentration consequently increases.
Hence, volume changes are an
artifact in measurements of TM motion. However, in the pH range of interest (pH <
37
4.5) in this experiment, there is less than 10% decrease in the thickness. The most
significant variations in volume change occur near pH 5 (Freeman et al., 1997), which
is above the pH level where we see significant variations in motion in the pH titration
experiment.
38
Chapter 5
Conclusions
This thesis presented a novel technique, in which we applied atomic force sensing to
the TM. We discovered motion on the surface of the TM caused by voltage application
delivered to the TM through microfabricated electrodes. The experiments included
in the results section showed that:
1) motion is on the order of nanometers and occurs as a result of AC voltage
application on the surface of the TM.
2) motion varies with changes in bath pH.
3) the TM moves like a seesaw with a node at the midpoint between the electrodes.
4) the pKa of the TM lies between 3.96 and 4.07 based on the 7r phase shift that
occurs between these two points in the pH experiments
Based on these findings, the next step is to develop a model to explain the source
of the induced TM motion. A model will not only uncover the values for the gel
model parameters (cf and r), but will also uncover any physiological relevance to the
induced motion. More specifically, a model of the motion will help to quantitatively
understand the effect of voltage on the TM, which will then help us gauge if electrically
induced motion is relevant in vivo. It is well known that the TM is surrounded by
small electric fields inside the cochlea (Fettiplace and Fuchs, 1999). The big picture
goal of this project that remains to be explored is to find out if the TM behaves
dynamically in the live cochlea.
39
40
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