Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Electrical, Computer & Energy Engineering

First Advisor

Robert McLeod

Second Advisor

Juliet Gopinath

Third Advisor

Jay Stockley

Fourth Advisor

Stephanie J. Bryant

Fifth Advisor

Carol Cogswell

Abstract

This research explores two applications that both require the ability to dynamically and efficiently redirect a laser to a volume of focal points in a sample. In the first application, a fabrication technique is presented to microlithographically-patterned three dimensional cellular structures in polymer hydrogels, with a particular focus on patterning C2C12 cells in linear arrangements to study the formation of muscle fibers. By exploiting the precision and control of microlithography to fabricate artificial tissues, this research aims to develop and demonstrate use of a tool that can be used to answer fundamental questions of developmental cell biology which cannot be addressed with existing randomly arranged 3D tissue scaffolds or 2D plated cells. In the second application a spatial light modulator (SLM) based microscope is presented that offers a new optical method of stimulating and monitoring interconnected cells in 3D environments. By non-invasively stimulating and imaging such 3D arrangements including artificial, ex vivo or in vivo tissue volumes, this research further aims to extend understanding of cellular interconnectivity, particularly in neural networks of brain slices. This highly interdisciplinary work was completed in collaboration with a diverse group of active international collaborators from the fields of micromanipulation, microlithography, cellular biology, and neuroscience.

The tissue engineering work presented in this thesis merges holographic optical tweezers for cell positioning with step-and-repeat 3D additive manufacturing to fabricate complex polymer microstructures of arbitrary scale containing internal cellular arrangements organized with micron-scale precision. These polymer micro-scaffolds support the cellular network and also provide channels for nutrient flow and directed cell growth. This research is the foundation of a new discipline of live cell lithography, which is used to enable biologists to study cell to cell signaling and cellular growth as a function of 3D cellular positioning in an environment that more realistically represents living tissue.

The second portion of the research extends the understanding of 3D cell networks using optogenetics. The project aims to develop a spatial light modulator (SLM) based microscope to enable optical monitoring and manipulation of the activity of neuronal ensembles, in vitro and in vivo. The outcome is a compact commercially available microscope that enables fast, 3D imaging and photoactivation of neurons. This can be used for imaging intact neural network activity, optical manipulation of neuronal firing, functional mapping of brain connectivity, investigating neurovascular coupling, and assaying neuronal activity in animal models of brain disease.

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