Transcript Final Poster

Determination of Intercellular Calcium Concentrations in Cardiac Myocytes Using
Fluorescence and a Single Fiber Optic Method
Paul Clark, Martin Garcia, Chris Gorga, John Ling, Jordan LoRegio
Sponsor: Dr. Franz Baudenbacher
INTRODUCTION
RESULTS (cont…)
EXPERIMENTAL SETUP
DEVICE FABRICATION
A
Figure 1: X-rod Emission and Excitation Spectra. Also
include in the figure above is the wavelength at which the
laser and filter are set.
C
Figure 2: Standard Steps of Soft Lithography. The basic
soft lithography used in fabricating the PDMS device
integrated with an optical fiber, allowing for real-time data
collection. Once plasma bonded to the glass slide the device
can capture cells up to 10 microns in diameter.
Proof of principle (i.e. changes in the magnitude of
fluorescence can be quantified using the integrated
optical fiber method) was achieved using a simple
single channel device and fluoroscein solutions of
varying concentrations. Figures 6a–6d, below, show
an increase in the magnitude of the voltage changes
as fluorescein concentration is increased.
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3.5
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2.5
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1.5
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2.5
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0
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40
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20
40
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100
Time (Seconds)
Time (Seconds)
Figure
6b:
10µM
Fluoroscein Solution. The
yield is approximately a
1.75V increase in voltage.
Figure 6a: 1µM Fluoroscein
Solution.
The
yield
is
approximately
a
0.4V
increase in voltage.
OBJECTIVES
o Design and fabricate a microfluidic device to
measure the calcium flux of a single cardiac
myocyte in real time as it contracts.
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CONCLUSIONS
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2.9
EXPERIMENTAL SETUP
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4.5
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5
Voltage (V)
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2.5
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1.5
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4
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2
o A microfluidic device has been developed allowing
for the quantification of the magnitude of
fluorescence using optical fibers as the detection
method.
o A second device has been developed that should
allow for the capture and excitation of single
cardiac myocytes.
o Electrodes specific to this device must be designed
for better alignment and larger platform for the
device.
o A better hole punching and fiber insertion
technique must be developed to prevent channel
collapse.
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0.5
0
0
0
Figure 4b: Data acquisition
setup. Shown here is the
final setup with the device
mounted on the microscope
ready for data acquisition.
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Figure 8b: Proper Device,
Electrode Alignment. At
right is the proper alignment
of the electrodes over the
cell loading area. Note the
optical fiber just to the upper
right as well as the collapsed
channels, later found to be
due to a damaged master.
RESULTS
Figure
4a:
Completed
device setup. Shown here is
the
completed
device,
mounted on the electrodes,
and clamped into place.
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Figure 8a: Loading of Live
Myocytes. At the left three
cardiac
myocytes
are
experiencing suction in an
attempt to load them via the
channel on the left of the
screen. A a collapsed
channel
prevented
a
successful loading.
Figure 5: Overall Schematic of Experimental Setup. The
system is capable of simultaneous dye excitement and
fluorescence reading. This is possible because of the optical
properties of X-rod couple with the dichroic filters. The PMT
(photomultiplier) amplifies and converts the flourescence
signal into a voltage which can be collected using LabView.
DEVICE SPECIFICATIONS
o Design and fabricate a microfluidic device showing
calcium flux can be quantified using an optical
detection method.
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Figure 7. Fluoroscein as Correlated to Voltage Increase.
The above trendline shows that the magnitude of voltage
increases linearly with increasing fluoroscein concentration.
B
Figure 3: PDMS device for trapping cardiac myocytes.
Note the cell inlet (A), main suction channel and debris
reservoir (B), fine suction channel (C), and cell chamber (D),
where the electrodes will be placed for excitation.
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[Fluoroscein] (µM)
Voltage (V)
A microfluidic platform was chosen to isolate single
cardiac myocytes for data acquisition via an
integrated optical fiber. The cells are died with the
fluorescent dye X-rod (emission and excitation
spectra below), which fluoresces when exposed to
calcium. Through electrical pulses the trapped cell is
coaxed into releasing calcium for its sER (smooth
endoplasmic reticulum), thus exciting the X-rod and
showing a change in fluorescence intensity.
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2.5
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1.5
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0.5
0
0
Voltage (V)
The development of a method for the study of
calcium signaling between cardiac myocytes will
lead to a great understanding of many cellular
processes, most notably contractile force. Once
quantified, the calcium flux data (for individual
myocytes) will be equated to a force, thus creating a
normal line equating calcium concentration to
contractile force.
D
Voltage (V)
Heart contraction depends on the coordination of the
electrical signals created by the individual cardiac
myocyte. Extracellular calcium flux allows cells to
communicate
with
neighboring
cells,
thus
propagating the electrical signal which was initiated
in the sinoatrial (SA) node.
Volatage (V)
[Fluoroscein] correlated to Voltage
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30
40
50
60
70
80
Time (Seconds)
Figure
6c:
15µM
Fluoroscein Solution. The
yield is approximately a 2.5V
increase in voltage.
0
10
20
30
40
50
60
Time (Seconds)
Figure
6d:
25µM
Fluoroscein Solution. The
yield is approximately a 4V
increase in voltage.
ACKNOWLEDGMENTS
This work would not have been possible without the advice of
our advisor, Dr. Baudenbacher or the help of graduate students
Tobias Meyer, David Schaffer, and Raghav Venkataraman.
This work was supported by funding from the Vanderbilt
Institute for Biosystems Research and Education (VIIBRE).