1. 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 Raghav Venkataraman, Tobias Meyer, David Schaffer Sponsor: Dr. Franz Baudenbacher 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 (in this case from cell to cell through the extracellular matrix) in order to propagate contraction through the heart. After the initial electrical stimulus provided by the sinoatrial (SA) node the individual cardiac myocytes release calcium, from intracellular stores such as the sarcoplasmic reticulum as they contract in order to signal to neighboring cells to do likewise. Calcium signaling is therefore 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. Thus allowing for a better understanding of how the healthy heart should behave. The microfluidic platform and digital system for data acquisition present allow for the collection of data regarding the change in magnitude of calcium flux as the force of myocyte contraction is modulated. INTRODUCTION The objective of this study is to measure the calcium concentration of single cardiac myocytes using a fluorescent dye as an indicator of intercellular concentration and a single fiber optic system to provide both excitation and emission paths. OBJECTIVE METHODS SUMMARY RESULTS Prior to measuring the calcium concentrations of cardiac myocytes, it was necessary to determine the range of fluorescence concentrations that could be detected by our photomultiplier tube (PMT). Fluorescein sodium salt was chosen as the initial fluorophore because of its common excitation and emission wavelengths. In order to measure the limit of detection of the PMT, small concentrations of fluorescein were run through a preliminary device with an embedded optical fiber. The channels were excited by a mercury lamp mounted within an inverting microscope. Extremely low concentrations were run through the device while a LabView program measured changes in voltage output. Figures 4 through 7 below represent a graph of changes in voltage as a function of time with the sudden increase in voltage representing the presence of fluorescein. Figure 4 shows the change in voltage as function of time using a 1 µM fluorescein solution. A clear voltage increase of approximately 1.5V represents the injection of fluoresecein. This represents the minimum concentration that was able to be detected by our PMT. Figure 5 and figure 6 show the same graphical representation for concentrations of 20 µM and 30 µM, respectively. The injection of fluorescein is represented by a gradual voltage increase 3.5V. Figure 7 shows the change in voltage for a fluorescein solution with a concentration of 1mM. The increase seen here is still of a 3.5 V magnitude, but the rate of increase is much larger. However, the rate of increase could either be a factor of concentration or injection speed. Because figures 5 through 7 all show voltage increases of the same magnitude it can be assumed that this displays a saturation of the PMT, and concentration on the micro level are more appropriate for useful data collection. Figure 8 shows the distortion that occurs when an extremely high concentration solution is passed through the device. To achieve a small compact setup that is easily fabricated, a PDMS device was produced using soft lithography techniques combined with internal fiber optics to produce real-time data collection. The cells being examined are single cardiac myocytes approximately 10 um in length. These cells increase internal calcium concentrations when external electrical stimuli is applied. The device is made up of an inlet, outlet, suction line, cell chamber, and all connection tubing as seen in figure 1. This fabrication consists of creating a reusable silicon master with harden SU-8 2025 microstructures using standard fabrication techniques as seen in figure 2. After master creation the PDMS device was created using a 10-1 ratio of monomer and curing agent respectively. Before application of the PDMS mix to the silicon master proper fiber placement must be achieved. This was done by carefully placing the fiber optic over the cell chamber using a micromanipulator and a 500 micron fiber. Once the fiber was placed the PDMS was poured and set in an oven till hardening occurred. After completely curing the PDMS device is removed from the master and electrical wires are placed on opposite sides of the cell chamber to properly excite the trapped cells and after this the device is plasma bonded to a glass slide so that testing can start. Testing begins with the induction of single cardiac cells into the transport channel and suction from the inlet to outlet. Once abundant cells are present a cardiac cells is pulled into the cell chamber on top of the previously placed electrodes. After placement any cells not in the cell chamber are flushed then a Xrod solution is inserted into the device so that the cardiac cells internally absorbs the dye. After ample absorption time the dye is removed and testing begins on the cell. Testing is performed using a device capable of simultaneous dye excitement and absorbance readings. This is made possible by both the excitement properties of the specific dye as well as integrated dichroic filters used to filter all but specific wavelengths out before PMT (Photomultiplier) tubes amplify and convert excitation to voltage readings. This setup can be seen in Figure 3. This voltage is then collected using a National Instruments DAQ assistant combined with a specifically designed lab-view program that displays and logs calcium concentration at a rate of 1000 Hz Figure 3: Excitation and Sensing setup in Integrated device. As seen above in image (A) the PMT, converter box (B), and computer are in series to filter and prepare data. Contrary to image (A) our setup does not contain a immersion microscopic as well as internally containing the excitation equipment (shutter, diffuser, filter, and dichroic mirror shown separate in the image above Two different microfluidic devices have been designed and constructed. The first device was used to determine the concentration of fluorosein to be used. The second device isolates a single cardiac myocyte and will allows fluorescence of a cardiac myocyte to be measured.. A concentration of 1e-6 M fluorosein was determined to be within the detectable range of the PMT box. The higher concentrations that were producing similar voltage jumps were saturating the box. The 1e-6 M produced a smaller voltage jump thus proving it is within the detectable range. It is this concentration of fluorosein that will be used in the final design. Integrating a fiber into the second design is the current goal. Once this is achieved then data using a cardiac myocyte can be collected. Eventually electrodes will be incorporated into the second device in order to induce contraction in the single cardiac myocyte. RESULTS (cont.) METHODS (cont.) Figure 1: PDMS device for trapping cardiac myocytes. Device shown with insert port (A), output port (B), suction channel (C), and cell chamber (D). The electrode inserts will be placed at the two ends of the cell chamber (D). A C B D Figure 2: Standard Steps of Soft Lithography. Steps of fabrication in order of photoresist application, exposure, and development followed by PDMS pouring and eventually plasma bonding of PDMS device to glass substrate. Image A Image B REFERENCES Sipido, Karin R., Callewaert, Geert (1995). How to measure intracellular [Ca 2+] in single cardiac cells with fura -2 or indo-1. Cardiovascular Research, 29, 717-726. Negative Tone Photoresist Formulations 2002-2005. Micro Chem Website, www.microchem.com.www.microchem.com Min-Hsien Wu, Haoyuan Cai, Xia Xu, Jill P.G. Urban, Zhan-Feng Cui, and Zheng Cui. A SU-8/PDMS Hybrid Microfluidic Device with Integrated Optical Fibers for Online Monitoring of Lactate. Biomedical Microdevices 7:4, 323 ミ 329, 2005. Fura-2 and Indo-1 Ratiometric Calcium Indicators. Molecular Probes, Invitrogen Detection Technologies. June 21, 2005. Fluorescein Sodium Salt Product Information. Sigma-Adrich Website, www.sigma-adrich.com. Connector and Connector Adapter Options. Ocean Optics Inc., www.oceanoptics.com/products/connectors.asp. SILICA/SILICA Optical Fiber, High -OH, UV Enhanced. Polymicro Technologies, LLC, www.polymicro.com www.sigma-adrich.com www.oceanoptics.com/products/connectors.aspwww.polymicro.com ACKNOWLEDGEMENTS This work was supported by funding from the Vanderbilt Institute for Biosystems Research and Education (VIIBRE). Figure 4: Fluoresecin concentration of 1µM Figure 5 (left) and Figure 6 (right): Fluorescein concentrations of 20 µM and 30 µM, respectively Figure 7 (left) and Figure 8 (right): Fluorescein concentrations of 1 mM and 6.6 mM, respectively Using lethargic cardiac myocytes a single cell was successfully isolated on two separate occasions. The cells were placed in the well of the PDMS device used for trapping the cells. A single cell was then suctioned into an isolation chamber. No dye was added to the cell, and no testing was done on the trapped cells. This process was completed to simply test the effectiveness of the device in trapping a single cardiac myocyte cell. Electrodes and an optical fiber need to be incorporated before any testing can be done.