Fluorescent Detection Fluorescent Detection using Optical Fibers using Optical Fibers with Cardiac Myocyteswith Cardiac MyocytesPaul ClarkPaul ClarkMartin GarciaMartin GarciaChris GorgaChris GorgaJohn Ling IIIJohn Ling IIIGiordano Lo RegioGiordano Lo Regio

ThesisThesis Device that is more Device that is more

cost efficient. cost efficient. Incorporate optical Incorporate optical

fibers into an existing fibers into an existing BioMEMS deviceBioMEMS device

The optical fiber or The optical fiber or fibers will detect the fibers will detect the fluorescence of fluorescence of calcium in contractile calcium in contractile cardiac myocyte cellscardiac myocyte cells

This design can This design can potentially be used to potentially be used to measure fluorescence measure fluorescence of any ion of interestof any ion of interest

QuestionsQuestions Optical FibersOptical Fibers

– What is the minimum What is the minimum fiber diameter to fiber diameter to produce accurate and produce accurate and differentiable results?differentiable results?

– Will a single fiber setup Will a single fiber setup work?work?

How successfully can How successfully can we integrate an optical we integrate an optical fiber into our PDMS fiber into our PDMS device?device?

How adaptable this How adaptable this device will be for other device will be for other ions?ions?

BackgroundBackground Completed micro fluidic Completed micro fluidic

device already known to device already known to successfully hold cardiac successfully hold cardiac myocytes. myocytes.

Online detection of Online detection of calcium using chemical calcium using chemical analysisanalysis

Already have large scale Already have large scale design of this process design of this process using inverted microscope using inverted microscope and multiple cell cultures.and multiple cell cultures.

Fura-2 dye specificationFura-2 dye specification Pre-designed Photo Pre-designed Photo

Multiplier Tube (PMT).Multiplier Tube (PMT).

ObjectivesObjectives Modify micro fluidic Modify micro fluidic

device to accept device to accept optical fiber.optical fiber.– Change consistency of Change consistency of

PDMSPDMS– Find correct positioning Find correct positioning

to achieve maximum to achieve maximum output signaloutput signal

Integrate small scale Integrate small scale fiber optics with PMTfiber optics with PMT– Adjust to smaller signalAdjust to smaller signal– Increase sensitivityIncrease sensitivity– Design LabView module Design LabView module

to analyze, graph, and to analyze, graph, and store input signalstore input signal

ImportanceImportance Provide integrated Provide integrated

system to quantify a system to quantify a host of intracellular host of intracellular moleculesmolecules– Charged IonsCharged Ions– Intra-membrane Intra-membrane

ProteinsProteins Could be used to Could be used to

examine intracellular examine intracellular molecules in several molecules in several single cells (not single cells (not exclusively myocytes)exclusively myocytes)

Social/Economic Social/Economic ImpactImpact Compact device Compact device

marketed to academic marketed to academic research institutionsresearch institutions

Alleviate start up costs Alleviate start up costs for smaller institutionsfor smaller institutions

More time and money More time and money devoted to experiment devoted to experiment rather than device rather than device developmentdevelopment

Focus on study of cell Focus on study of cell and its physiologyand its physiology

StatusStatus Optics of the PMT box are being studiedOptics of the PMT box are being studied

– Magnitude of the signal Magnitude of the signal – Signal amplification into Labview for data Signal amplification into Labview for data

aquisitionaquisition– Will allow for the addition of the chopperWill 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 A master for the PDMS micro-fluidic device

has been designedhas been designed– Waiting on mask to be castWaiting on mask to be cast

Outlined next 3 weeksOutlined next 3 weeks

Photomask Photomask SpecificationsSpecifications Channels (3) are 200 microns wideChannels (3) are 200 microns wide

– Dictated by fiber sizeDictated by fiber size Channels are spaced 100 microns apartChannels are spaced 100 microns apart Optical fibers to be incorporated are 250, Optical fibers to be incorporated are 250,

150, 100, and 50 microns in diameter150, 100, and 50 microns in diameter

Mask DesignMask Design

Optical Fibers

Master FabricationMaster Fabrication SU-8 is spun onto a silicon SU-8 is spun onto a silicon

substratesubstrate– SU-8 2025 is usedSU-8 2025 is used

Master is spun for 30sec at Master is spun for 30sec at 1750rpsec1750rpsec– Gives desired thickness of 50 micronsGives desired thickness of 50 microns

Master is then curedMaster is then cured

PDMS casting and PDMS casting and Optical Fiber IntegrationOptical Fiber Integration Three micromanipulators are used to align the optical fibers Three micromanipulators are used to align the optical fibers

over the channelsover the channels PDMS is poured master and optical fibers PDMS is poured master and optical fibers

– Thickness can be altered each timeThickness can be altered each time– Placed in a vacuum to remove air bubblesPlaced in a vacuum to remove air bubbles

PDMS is then curedPDMS is then cured– Hardness is determined by time curedHardness is determined by time cured– PDMS is peeled off and placed on a microscope slidePDMS is peeled off and placed on a microscope slide

ConclusionsConclusions Separately the two parts are not difficult to designSeparately the two parts are not difficult to design

– Initial designs for both have been agreed upon Initial designs for both have been agreed upon – MEMS fabrication is very simple and easily repeatableMEMS fabrication is very simple and easily repeatable

Difficulty is in the integration of the optical fibers Difficulty is in the integration of the optical fibers with acceptable output signal magnitudewith acceptable output signal magnitude– Sensitivity is highly correlated to fiber sizeSensitivity is highly correlated to fiber size

Want to use smallest fiber possibleWant to use smallest fiber possible In the near future…In the near future…

– Fabrication of masterFabrication of master– Casting of initial PDMS device integrated with optical fibersCasting of initial PDMS device integrated with optical fibers– Begin testing just fluorescent die in channelsBegin testing just fluorescent die in channels

If successful begin to plan for used of smaller fibers and If successful begin to plan for used of smaller fibers and possibly smaller channels (width)possibly smaller channels (width)