Date of Award

Fall 2007

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Electrical & Computer Engineering

Program/Concentration

Electrical Engineering

Committee Director

Karl Schoenbach

Committee Director

Richard Nuccitelli

Committee Member

Helmut Baumgart

Committee Member

Ravindra P. Joshi

Call Number for Print

Special Collections LD4331.E55 S525 2007

Abstract

I have fabricated a chamber which enables the application of Nanosecond Pulsed Electric Fields (nsPEFs) to living cells while monitoring their real time fluorescence with an inverted microscope. The No. I thickness (150µm nominal) coverslip glass substrate used for fabrication allows the use of high numerical aperture oil immersion objectives for imaging intracellular organelles. Electrodes are fabricated by first evaporating a chromium adhesion layer, followed by a nickel seed layer. Subsequently, an ultra-thick positive photoresist is applied and patterned to create a 30µm thick electroplating mold. The electrodes are then electrodeposited into the mold from a nickel sulfamate plating bath. These electrodes are gold plated to ensure biological inertness. The process results in 25µm thick gold-plated electrodes with near vertical side walls deposited directly onto the glass coverslip with no glue layer. The electrode gap distance is 100µm which enables electric fields of up to I00kV/cm to be applied with a solid state switched Blumlein line pulse generator used in the microscope pulsing apparatus. I have applied nsPEF's to B16-FI0 Murine Melanoma cells and have recorded real time Calcium change using the indicator Fluo-4 AM. These preliminary experiments were conducted at a physiological temperature of 37°C and suggest B16 cells recover from intracellular Calcium release within minutes. In the development of this process I have minimized fabrication costs while selecting steps to maximize ease of processing. Several innovations in processing are discussed which allow semiconductor-style fabrication involving thin film deposition, photolithography, and electrodeposition to be performed outside of a conventional cleanroom while yielding acceptable results. Once fabricated,

I conducted the first biological experiments with this chamber by studying the changes in intracellular Ca2+ in B16 melanoma cells in response to Nanosecond Pulsed Electric Fields. The main findings emerging from this work are: I) Intracellular Ca2+ increases immediately upon exposure to nsPEF as low as 30 kV/cm; 2) Larger imposed fields in the range of 30-100 kV/cm result in a larger Ca2+ increase; 3) The recovery to normal Ca2+ levels is highly temperature dependent with recovery time at the physiological temperature of 37°C averaging one minute compared to greater than 10 minutes at room temperature; 4) The role of extracellular Ca2+ influx was examined by measuring the Ca2+ increase in the absence of extracellular Ca2+. This revealed that intracellular Ca2+ release is 45% of the total Ca2+ flux; and 5) The magnitude of Ca2+ increase is dependent upon the pulse frequency.

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DOI

10.25777/4fp9-xg48

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