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Vanderbilt University
Department of Biomedical Engineering
Determination of Intercellular Calcium
Concentrations in Cardiac Myocytes
Using Fluorescence and a Single Fiber
Date Performed: Spring 2007
Date Reported: April 24, 2007
Reported by:
Paul Clark
Martin Garcia
Chris Gorga
John Ling
Jordan LoRegio
Abstract
In order to understand the dynamics of myocyte contraction it is important to
examine calcium signaling. Developing methods for studying calcium flux by cardiac
myocytes should allow for a greater understanding of many cellular processes within
cardiac myocytes, most notably contractile force. Quantifying the magnitude of the
calcium flux by cardiac myocytes should allow for the development of a relationship
between said calcium flux and the contractile force of individual myocytes. Examining
cells at a point in time during contraction may not allow for a full understanding of the
system, however it will allow for the development of a “normal line” for the contractile
force and magnitude of calcium flux. A polydimethylsiloxane device was made using
soft lithography techniques that was easy to fabricate, transparent, and biologically inert.
During the course of the project two distinct devices were made to facilitate testing. The
first device was a simple three-channel device testing the excitability of a fluorescent dye
and the criteria for proper data collection and the second device was a single layer PDMS
nanophysiometer used for the trapping of cardiac myocytes. Each of these devices were
fitted with a 200µm optical fiber and tested using a Lab View software program. The
final device was equipped with electrodes used to excite the cells during testing. The
goal of the first portion was achieved by embedding an optical fiber into the device and
then used to detect changes in magnitude of fluorescence. From the results, it was found
that a single cardiac myocyte in a newly fabricated device could be induced to contract
via electrical stimulation, and data could optically record change in fluorescence. In
conclusion, the design gave proof of concept for the idea that the transient calcium of a
contracting cardiac myocyte can be directly measured using a bioMEMs device
incorporating a single optical fiber.
Introduction
Normal contraction of the heart depends on many factors from the complicated
coordination of electrical stimulus to the communication between neighboring cells. Just
as in many other cellular processes, cardiac myocytes utilize calcium flux. However, in
the case of cardiac myocytes, cells are directly connected to each other via intercalated
discs. These intercalated discs form gap junctions between the cytoplasm’s of adjacent
cells, thus propagating contraction throughout the heart1. Cardiac myocytes are also
myogenic, meaning that they are self-excitable and without external input, will contract
rhythmically at a steady rate set by the autonomic nervous system2. Initiation of
contraction is provided by the
sinoatrial (SA) node due to the
release of calcium from intracellular
stores such as the sarcoplasmic
reticulum. The SA node
immediately causes contraction of
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the atria while the impulses to the
ventricles is slightly delayed by the
atrioventricular node. Once the atria
have been emptied to the ventricles,
Figure 1 - Diagram of the Heart and Essential
Nodes of Electrical Activity
the impulse passes to the bundle of
His which denotes separate paths for each ventricle. Finally, the Purkinje fibers, which
line the walls of both ventricles, rapidly conduct the impulse to cause the myocytes to
contract form the bottom up, thus maximizing ejection of blood (see Figure 1).
The focus of this project is to examine calcium signaling in order to develop a key
to understanding the dynamics of myocyte contraction. Developing methods for studying
calcium flux by cardiac myocytes should allow for a greater understanding of many
cellular processes within cardiac myocytes, most notably contractile force. Quantifying
the magnitude of the calcium flux by cardiac myocytes should allow for the development
of a relationship between said calcium flux and the contractile force of individual
myocytes. Examining cells at a point in time during contraction may not allow for a full
understanding of the system, however it will allow for the development of a “normal
line” for the contractile force and magnitude of calcium flux.
Several studies have examined different methods for accurately determining the
contractility of the heart by measurement of calcium concentration and/or flux. These
methods can range from simply measuring calcium concentrations using chemical
methods to a host of different optical methods. This paper will limit background
discussion to topics strictly related to optical detection. One previous study attempted to
measure the calcium concentration in single cardiac myocytes of brown mice3. The
experimental setup included mounting single myocytes, which had already been injected
with a fluorescing agent (in this case fura-2), on an inverted microscope. Once cells were
properly positioned, the intracellular fura-2 was excited by a mercury/xenon lamp and
passed through a filter specific to fura-2 excitation (360nm). Emission was collected
through another, different filter that was limited to fura-2 emission wavelengths (500nm).
The emitted light then entered a photomultiplier tube to be converted to a corresponding
voltage. Throughout this process, the myocytes were contracted mechanically using field
stimulation. Other studies have attempted to use a CCD camera with an intensifier rather
than a photomultiplier tube to measure emitted fluorescence. In the case of this study it
was necessary to attach a chopper and shutter to a Xenon arc lamp in order to alternate
between light frequencies (see Figure 2).
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Figure 2 - Previous Device Containing Lamp, Mirror Chopper, and Inverted Microscope
Most previous studies have presented rather complicated and material intense
methods to measure intracellular calcium in single myocytes. Both of the studies
mentioned require an inverted microscope with an internally mounted Hg/Xe lamp. The
first study mentioned also mechanically stimulates the cardiac myocytes which is an
unfavorable method because it forces cells to contract at a set length, which maybe more
or less than the contraction range of the individual cell.
Our design team, with the help of Dr. Franz Baudenbacher and several graduate
students, is hoping to develop a less complicated and more accurate method for
measuring calcium concentrations in single cardiac myocytes. The proposed system will
include a microfluidic device to trap and stimulate single cells and an optical box that
will provide excitation and measure emission. Figure 3 below displays a microfluidic
device that will be composed of polydimethylsiloxane (PDMS) from both the side
(Figure 3A) and enlarged top (Figure 3B). Figure 3B shows the reservoir where the
myocytes will be injected to the device after first being soaked in a fluorescing agent and
then pulled through the main channel via the drain. The
smaller trap control pulls a cell into the small trap area
(120µm X 30µm). Figure 3C displays enlarged view of
the placement of the optical fiber in the microfluidics
device. Rather than using a mechanical stage to contract
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the myocytes, electrodes will be placed within the trap to
electronically stimulate the myocyte while excitation
and emission is allowed to pass through the fiber and
back to the optical box.
Figure 4 shows the optical box from the side
(Figure 4A) and enlarged top (Figure 4B). Figure 4A
Figure 3 - Diagram of
microfluidics device with
side view (A), enlarged top
view (B) and fiber capture
volume (C)
shows placement of the optical fiber in the optical box
that provides a pathway for both excitation and emission
wavelengths. Figure 4B shows the basic
inner workings of the optical box with a
green diode laser and a photomultiplier
tube (PMT) connected to an optical cube
containing a dichroic mirror. Power will
be provided to both the laser and the PMT
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while data output is obtained from the
PMT. Figure 4C displays the two optical
pathways that are present within the
aforementioned cube. Laser light passes
Figure 4 - Diagram of Optical box with
side view (A), internal view (B), and
dichroic mirror (C)
from the laser to the dichroic mirror where it is directed into the fiber and ultimately to
the individual myocyte (Figure 3C). Simultaneously, emitted light passes from the
myocyte through the optical fiber and bypasses the dichroic mirror. Before entering the
dichroic
mirror, the
emitted light
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first passes
through a
filter
specific to
Figure 5 - Excitation Emission Spectra of X-Rhodamine including the
laser (yellow) and high pass emission filter (red)
the
fluorescing
agent. The PMT then converts the relative fluorescent value to a voltage and provides a
data output to a computer. The fluorescing agent chosen for this system is X-Rhodamine
(XRITC), which is a water-soluble dye that emits fluorescent light in the presence of
calcium. XRITC has excitation and emission peaks at 580nm and 605nm, respectively.
Figure 5 displays fluorescent spectra of XRITC with the yellow line denoting a laser
source and the red line denoting the presence of a high pass emission filter.
The system described contains much fewer elements than previous systems while
still providing an accurate method of quantifying intracellular calcium concentrations.
By removing the need for an inverted microscope, a mechanical stimulating stage, or an
additional excitation filter (lasers only emit one wavelength), the system becomes more
affordable and compact.
Methods
As per the requirements above, a device was made that was easy to fabricate,
transparent, and biologically inert. The natural
choice was to use a polydimethylsiloxane
(PDMS) device that meets all the above
specifications and is very quick to make (<24
hrs design to final). The PDMS device was
fabricated using soft-lithography. The softlithography process used consisted of many
steps as can be seen in Figure 6. The
fabrication of the device started with a good
quality master. A three-inch silicon dioxide
wafer was used as the master substrate (Figure
Figure 6 - Diagram of master
processing4
6A). The surface features on the master were
crafted out of the negative photoresist SU-8 20254. SU-8 2025 was used due to
availability and its ability to produce features high enough to form the channels required
in our device (40 to 50 µm). The process started by spinning the SU-8 onto the master at
2500 rpm for 30 seconds when an even coating height was achieved (Figure 6B). After
baking (Figure 6C) the SU-8 was exposed (Figure 6D) to ≈120 mW of UV light that
initiated cross-linking in non-masked regions. A CAD created photographic mask was
used during the exposure stage. A Post-Exposure bake (Figure 6E) was performed prior
to the development. Development (Figure 6F) consisted of various washes using
Acetone, Isopropyl Alcohol, and SU-8 developer made by MicroChem. Once developed
the master was ready to be used to construct the final PDMS device4.
The PDMS processing steps were quite simple compared to that of the master. To
produce the PDMS solution a monomer solution was mixed with a curing agent in a 10:1
ration respectively. This ratio produced a stable but flexible device ideal for inserting an
optical fiber. A micromanipulator was used in combination with other devices for
holding the fiber in place while pouring the PDMS onto the device. We later found that
placing the fiber onto the master structures scratched or removed the SU-8 structures on
the master. To combat this, the fiber was placed above the channels by poking holes into
the device after it was cured. The device was cured by placing it in a warm oven at 65o C
for approximately one hour4. After curing, the PDMS device was removed from the
silicon and any necessary holes were punched in the device. The device was then plasma
bonded to a glass substrate to produce a water-tight seal. The device was then ready for
testing.
During the course
of the project two
Optical Fibers
distinct devices were
simple three-channel
3 mm
The first device was a
2 mm
made to facilitate testing.
200 um
40 um Deep
device testing the
excitability of a
fluorescent dye and the
Figure 6 - Mock-up mask of first device design (Not to Scale)
criteria for proper data collection. The three channels ran in parallel, each being 200
microns wide x 40 microns high. The channel length was approximately 2 mm long to
ensure proper fluid flow and mixing over the length of the channel. A mock up of this
design can be seen in Figure 6. To collect data a polymer fiber optic cable was placed
over the channels. Polymer was used due durability issues of glass during the baking and
plasma bonding processing steps of fabrication. Fluorescent detection was done in a twostage process. An inverting microscope, with an internal Hg/Xe Lamp was used as the
excitation source after being fitted with a specific band-pass filter (440nm  510nm).
Fluorescence collection was done using a fiber optic inserted on top of the fluid channels.
The dye used for detection was flourescein sodium salt. Flourescein is a pH sensitive
water-soluble dye with an excitation wavelength at 460nm2 and an emission wavelength
at 515 nm2. The emission energy from the flourescein was collected using the optical
box described in Figure 4. This device contained a photmultiplier tube (PMT) that
converted a fluorescence value into a corresponding voltage. The circuit diagram for the
PMT can be seen in Appendix A. The voltage from the PMT is then output to a LabView
USB DAQ assistant via a BNC cable. A LabView program was used to analyze and
record fluorescence data at a sampling rate of 1000Hz.
The second device created was a single layer PDMS nanophysiometer used for
the trapping of cardiac myocytes. The cardiac myocytes used were removed from a
profused murine heart and were still functional. The device can be seen in Figure 3B and
consists of an cell reservoir, a trap control, a drain, and a cell chamber. As well as a more
complex design this device also had a modified detection system. In place of the
inverting microscope above a single fiber optic acted as the excitation source, and also
was used to carry the emission energy back to the photomultiplier box as in the first
device. Therefore both the excitation and collection devices were housed in the optical
box mentioned above (Figure 4). This was made possible by the addition of a filter acting
much like a one way mirror so that only the emission wavelength was passed to the PMT.
The excitation photons were produced using a green laser with a frequency of 532nm. In
place of fluorescein a dye, X-Rhodamine (XRITC), was used for fluorescence detection.
This was due to the calcium binding properties of XRITC necessary for this experiment.
XRITC has a max excitation wavelength of 494 nm and a max emission wavelength of
518 nm6. In order for Ca2+ differentials to be observed the cardiac cells had to be
stimulated so that consistent contraction occurred. To accomplish this, electrodes made
of platinum were deposited onto a glass substrate. These electrodes were then matched
up with the cell chamber mentioned before so that an electrical potential could be created
across the cell. This design was not plasma bonded as before but was instead clamped
down so that an airtight seal was formed between the PDMS device and glass substrate
that the metal electrodes were deposited on. Stimulation of cardiac cell was to be carried
out at a frequency of 1Hz for 10 seconds while the laser was pulsed at intervals long
enough to observe the cardiac calcium transient. As in the above experiment the DAQ
assistant and labview were used for data analysis and storage.
Results
In order to show that an optical fiber could be used to detect fluorescence in
microfluidic devices a simple, one channel device was created with a single, 200µm
optical fiber embedded within the PDMS directly above the channel. Fluoroscein was
chosen due to its common excitation and emission wavelengths. Fluoroscein solutions of
varying concentrations (1, 10, 15, and 25µM respectively) were excited using a
mercury/xenon lamp mounted inside an inverting microscope. The signal was amplified
and converted form a fluorescence value to a voltage by the PMT box. LabView was
used to record and store the data as a voltage. The data could then be opened and
analyzed using excel.
The goal of the first portion of the experiment was to examine whether an optical
fiber could be embedded into a device and then used to detect changes in the magnitude
Voltage v. Time
Voltage v. Time
4 .5
4
3
3 .5
V o lta g e ( V )
Vo ltag e (V)
2.9
2.8
2.7
3
2 .5
2
1 .5
1
2.6
0 .5
0
2.5
0
10
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50
60
70
80
T im e ( S e c o n d s )
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0
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20
30
40
50
T im e (S e c o n d s )
Figure 7 - Voltage vs. Time graph of a
fluorescein concentration of 10μM, with a
jump in magnitude of approximately 0.4V
Figure 8 - Voltage vs. Time graph of
fluorescein concentration of 15mM yielded
an increase of 4µM
of fluorescence. Proving this
hypothesis to be true would yield a positive proof of principle. The change in magnitude
of fluorescence for each of the four fluoroscein solutions was found by subtracting the
baseline voltage created by the buffer solution from the final voltage create after the
injection of said fluoroscein solutions. The first concentration to be tested was the 1µM
solution, which yielded a jump from the baseline (2.53V) to 2.93V, a difference of 0.4V.
This data is shown graphically in Figure 7. This process was conducted for each of the
three other fluorescence concentrations, all of which showed a similar jump in from the
baseline voltage after the solution was injected into the buffer. The 10µM, 15µM, and
25µM solutions showed increases of 1.75, 2.5 (shown graphically in Figure 8) and 4V
respectively. These results are summarized in Table 1 below.
Results Summary
Concentration(µM)
Baseline Voltage(V)
Final Voltage(V)
Δvoltage(V)
1
10
15
25
2.53
1.5
1.5
1
2.93
3.25
4
5
0.4
1.75
2.5
4
Table 1 - Recorded voltages and the calculate jumps in voltage
Also noted during the course of conducting these experiments, is that the rate of
increase in voltage is much larger in the higher concentrations in the higher
concentrations. This could either be a direct result of the concentrations being so much
higher causing the maximum fluorescence to be detected at a quicker rate or it could be
due to the method used for flowing the fluorescein through the channel. Since the
fluorescein was being pushed through by hand using a simple syringe then the rate of the
injection of fluorescein into the microfluidic device could also have contributed to the
rate of change of the voltage. These
results prove that the hypothesis was
correct in stating that an optical fiber
can be embedded in a PDMS device
and used to detect changes in
fluorescence. In addition, these basic
experiments prove that the PMT was
able to detect minute changes in
Figure 9 - New entry for cells after reservoir
was cleaved
flourescein concentration within the device.
The goal of the second portion the investigation is to isolate a single cardiac
myocyte and in a newly fabricated device, induce contraction via electrical stimulation,
and optically record the change in fluorescence (of the XRITC which is excited by the
presence of calcium). The nanophysiometer described above in the methods was
suggested for use because of its compatibility with cardiac myocytes. Due to the small
size of the electrodes the device had to be modified in order to fit on the platform. This
resulted in the loss of the cell reservoir (new edge of device shown in Figure 9), which
caused for a partial collapse of the channel wall. This decrease in channel height led to
the channel being too small for the cells to fit through.
In order to alleviate this problem, the cutting of the device must begin on the
bottom side of the device before it is bound to the electrode. Therefore the device was
recast, cut, and realigned on the electrode in order to once again attempt to test on a
cardiac myocyte. However, upon mounting the fully assembled device on the inverted
microscope, it was discovered that the fine channels that are used for the control of the
suction had collapsed (see Figure 10). At first it was thought that this was only debris, but
attempts to flush the channels failed.
This led to the examination of the master
for defects. Here it was found the master
had been worn, and was useless. Due to
time limitations this was the final attempt
taken to load a single cardiac myocyte
into the device.
Figure 10 - Device mounted with collapsed
channels
Conclusion
This design project provided a proof of concept for the idea that the transient
calcium of a contracting cardiac myocyte can be directly measured using a bioMEMs
device incorporating a single optical fiber. A simple single channel microfluidic device
was developed allowing for the quantification of the magnitude of fluorescence using
optical fibers as the detection method. This provided the proof of concept for using an
optical fiber with a microfluidic device. A second device was developed that should
allow for the capture and excitation of single cardiac myocytes. Due to time and certain
design constraints data was not successfully collected for the second device.
Recommendations
The electrodes should be specifically designed for the particular nanophysiometer
bioMEMs device being used. Using electrodes not specifically designed for the chosen
device provided significant alignment problems that proved too difficult to overcome
without redesigning the device or electrodes. A more efficient and reliable method for
inserting a fiber into a device with such a small channel (120µm
30µm) needs to be
developed to significantly shorten the amount of time required for assembling one device.
Fitting a micrometer with tweezers or some other method of holding the fiber would
make it much easier to align the fiber over the channel under a microscope.
References
1. Cheung, J.Y, Tillotson, D.L., Yelamarty, R.V. Cytosolic Free Calcium Concentration
in Individual Cardiac Myocytes in Primary Culture. American Journal of
Physiology, 256: pp. 1120-1130. June 1989.
2. Sherwood, L. Chapter 9: Cardiac Physiology. Human Physiology: From Cells to
Systems Fifth Edition. Brooks/Cole, Belmont, CA. 2004.
3. Qun, Li., Fang, C.X., Nunn, J.M. Characterization of Cardiomyocyte ExcitationContraction Coupling in the FVB/N-C57BL/6 Intercrossed Brown Mice. Life
Sciences, 80(3): pp. 187-192. December 2006.
4. MicroChem. (n.d.). SU-8 2000. Retrieved April 23, 2007, from MicroChem:
http://www.microchem.com/products/pdf/SU-
5. Sigma-Aldrich. (n.d.). Fluorescein sodium salt. Retrieved March 15, 2007, from
Sigma-Aldrich: www.sigma-aldrich.com
6. Optical, O. (n.d.). Curve-o-matic. Retrieved March 22, 2007, from Omega Optical:
https://www.omegafilters.com/curvo2/index.php
7. Molecular Probes, Invitrogen Detection Technologies. Fura-2 and Indo-1 Ratiometric
Calcium Indicators. June 21, 2005.
8. Sigma-Adrich (n.d.). Fluorescein Sodium Salt Product Information. Retrieved March
22, 2007, from www.sigma-adrich.com
9. Ocean Optics Inc. (n.d.). Connector and Connector Adapter Options. Retrieved March
24, 2007 from www.oceanoptics.com/products/connectors.asp
10. Polymicro Technologies. SILICA/SILICA Optical Fiber, High -OH, UV Enhanced.
Retrieved Marh 24, 2007 from www.polymiro.com
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