Fluorescent Detection using Optical Fibers with Cardiac Myocytes Paul Clark Martin Garcia Chris Gorga John Ling III Giordano Lo Regio

Thesis Device that is more cost efficient. Device that is more cost efficient. Incorporate optical fibers into an existing BioMEMS device Incorporate optical fibers into an existing BioMEMS device The optical fiber or fibers will detect the fluorescence of calcium in contractile cardiac myocyte cells The optical fiber or fibers will detect the fluorescence of calcium in contractile cardiac myocyte cells This design can potentially be used to measure fluorescence of any ion of interest This design can potentially be used to measure fluorescence of any ion of interest

Questions Optical Fibers Optical Fibers –What is the minimum fiber diameter to produce accurate and differentiable results? –Will a single fiber setup work? How successfully can we integrate an optical fiber into our PDMS device? How successfully can we integrate an optical fiber into our PDMS device? How adaptable this device will be for other ions? How adaptable this device will be for other ions?

Background Completed micro fluidic device already known to successfully hold cardiac myocytes. Completed micro fluidic device already known to successfully hold cardiac myocytes. Online detection of calcium using chemical analysis Online detection of calcium using chemical analysis Already have large scale design of this process using inverted microscope and multiple cell cultures. Already have large scale design of this process using inverted microscope and multiple cell cultures. Fura-2 dye specification Fura-2 dye specification Pre-designed Photo Multiplier Tube (PMT). Pre-designed Photo Multiplier Tube (PMT).

Objectives Modify micro fluidic device to accept optical fiber. Modify micro fluidic device to accept optical fiber. –Change consistency of PDMS –Find correct positioning to achieve maximum output signal Integrate small scale fiber optics with PMT Integrate small scale fiber optics with PMT –Adjust to smaller signal –Increase sensitivity –Design LabView module to analyze, graph, and store input signal

Importance Provide integrated system to quantify a host of intracellular molecules Provide integrated system to quantify a host of intracellular molecules –Charged Ions –Intra-membrane Proteins Could be used to examine intracellular molecules in several single cells (not exclusively myocytes) Could be used to examine intracellular molecules in several single cells (not exclusively myocytes)

Social/Economic Impact Compact device marketed to academic research institutions Compact device marketed to academic research institutions Alleviate start up costs for smaller institutions Alleviate start up costs for smaller institutions More time and money devoted to experiment rather than device development More time and money devoted to experiment rather than device development Focus on study of cell and its physiology Focus on study of cell and its physiology

Status Optics of the PMT box are being studied Optics of the PMT box are being studied –Magnitude of the signal –Signal amplification into Labview for data aquisition –Will allow for the addition of the chopper Gives specific excitation frequency (kHz) Gives specific excitation frequency (kHz) A master for the PDMS micro-fluidic device has been designed A master for the PDMS micro-fluidic device has been designed –Waiting on mask to be cast Outlined next 3 weeks Outlined next 3 weeks

Photomask Specifications Channels (3) are 200 microns wide Channels (3) are 200 microns wide –Dictated by fiber size Channels are spaced 100 microns apart Channels are spaced 100 microns apart Optical fibers to be incorporated are 250, 150, 100, and 50 microns in diameter Optical fibers to be incorporated are 250, 150, 100, and 50 microns in diameter

Mask Design Optical Fibers

Master Fabrication SU-8 is spun onto a silicon substrate SU-8 is spun onto a silicon substrate –SU is used Master is spun for 30sec at 1750rpsec Master is spun for 30sec at 1750rpsec –Gives desired thickness of 50 microns Master is then cured Master is then cured

PDMS casting and Optical Fiber Integration Three micromanipulators are used to align the optical fibers over the channels Three micromanipulators are used to align the optical fibers over the channels PDMS is poured master and optical fibers PDMS is poured master and optical fibers –Thickness can be altered each time –Placed in a vacuum to remove air bubbles PDMS is then cured PDMS is then cured –Hardness is determined by time cured –PDMS is peeled off and placed on a microscope slide

Conclusions Separately the two parts are not difficult to design Separately the two parts are not difficult to design –Initial designs for both have been agreed upon –MEMS fabrication is very simple and easily repeatable Difficulty is in the integration of the optical fibers with acceptable output signal magnitude Difficulty is in the integration of the optical fibers with acceptable output signal magnitude –Sensitivity is highly correlated to fiber size Want to use smallest fiber possible Want to use smallest fiber possible In the near future… In the near future… –Fabrication of master –Casting of initial PDMS device integrated with optical fibers –Begin testing just fluorescent die in channels If successful begin to plan for used of smaller fibers and possibly smaller channels (width) If successful begin to plan for used of smaller fibers and possibly smaller channels (width)