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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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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). QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 20 30 40 50 60 70 80 T im e ( S e c o n d s ) 2.4 0 10 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 1 2 T U O M 5 2 5.2pF 2 R 3 C 3.4K M 1 F n 0 0 1 -15 7 R 6 R 7 C 0 4 4 U 0 OPA124PA AD711JR 1 1 R 5 1 3 B 1 2 U F n 0 0 5 2 1 0 R 6 2 C 3 5 6 2 F n 0 0 F 2 1 n 0 0 0 0 V 0 1 C -15V 1 B 7 FILTER OUT 2 8 1 C 7 8 0 5 1 F n 0 0 1 6 C +15V M 5 2 K 2 3 1 R - 8 R + PMT 4.7pF 4 C