Copyright by Sertan Kutal Gokce 2016
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Copyright by Sertan Kutal Gokce 2016 The Dissertation Committee for Sertan Kutal Gokce Certifies that this is the approved version of the following dissertation: Parallel and Serial Microfluidic Platforms for Femtosecond Laser Axotomy in Caenorhabditis elegans for Nerve Regeneration Studies Committee: Adela Ben-Yakar, Supervisor Mikhail A. Belkin Andrew Dunn Mark F. Hamilton Jon Pierce-Shimomura Preston S. Wilson Parallel and Serial Microfluidic Platforms for Femtosecond Laser Axotomy in Caenorhabditis elegans for Nerve Regeneration Studies by Sertan Kutal Gokce, B.S.; M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2016 Dedication Annem Arife Çay’a, memleketim Adana’ya ve Galatasaray’a. To my mom Arife Çay, my hometown Adana, and Galatasaray. Acknowledgements First, I would like to thank my advisor Dr. Adela Ben-Yakar for her guidance, encouragement, and financial support throughout my Ph.D. studies at The University of Texas at Austin. Her enthusiasm and passion for multidisciplinary research and neuroscience have inspired numerous great projects such as the ones described here. I would like to thank my committee members Dr. Mikhail Belkin, Dr. Andrew Dunn, Dr. Mark F. Hamilton, Dr. Jon Pierce-Shimomura, and Dr. Preston S. Wilson. They have been supportive throughout my Ph.D. study. In particular, my most sincere appreciation is extended to Dr. Jon Pierce-Shimomura and Dr. Preston S. Wilson for their help and guidance during my Ph.D. studies and their valuable advice for my future career. I wish to thank all my past and present fellow lab members for making this Ph.D. journey interesting and fun and for providing their support when I struggled. I would like to thank especially Dr. Onur Ferhanoglu, Dr. Sudip Mondal, Dr. N. Ghorashian, Travis Jarrell, Aubri Kottek, Dr. Sudip Mondal, Dr. Ryan Doonan, Dr. Ki Hyun Kim, Dr. Neil Everett, Evan Hegarty, Christopher Michael Martin, Dr. Frederic Bourgeois, Sam Aminfard, Dr. Ilan Gabay, Dr. Dan Eversole, Peisen Zhao, Kaushik Subramanian, and Dr. Fred Li. Next, I would like to thank all JPS lab members, especially Dr. Luisa Scott, Dr. Andres Vidal-Gadea, Sarah Nordquist, and Claire Celia Beron for always being helpful and listening to my problems regarding research. Next, I would like to thank Dr. Sydney Geissler, Anna Warden, and Roberto Ulises Cofresí for helping me with editing my dissertation and for answering my questions regarding biology. v My sincere appreciation is extended to my dear friends Dr. Onur Kacar, Hande Gerkus, Dr. Yiorgos Zalachoris, Elif Zeynep Iyriboz, Dr. Can Hankendi, Batur Isdiken, Dr. Sanjiv Shah, Funda Donmez, Onur Domanic, Gokhan Yildiz, and Quack’s Bakery crew. I want to thank them for making my life in Austin joyful and sharing my Ph.D. journey. I want to thank my good friends in Turkey, who shared my Ph.D. journey over a very long distance. I want to thank Denizcan Soner, Umut Toklu, Murat Atak, Sabit Sagir, Emre Aytekin, Baran Evliyaoglu, Can Yagli, Fidel Berber, Onder Ucar, Utku Ozkan, Onur Gokpinar, Ilker Gezer, Mert Pala, Arda Bahadir, and Sencer Ucar. Last, but never the least, I would like to declare my deepest appreciation and greatest gratitude to my father, Salih Gökçe, my wonderful mom, Arife Çay, my grandparents who always believed in me and wanted me to pursue higher education, Ali Şahin Çay and Hatice Çay, my super aunt, Mürüvvet Çay, my aunts, Sacide Çay and Ülviye Gürbüz, my uncle, Yasin Çay. I do not believe I would ever have been able to seek higher education without your endless support and love. I also wish to recognize the many people that have made this dissertation a reality. In addition, I apologize in advance for the many people who I forgot to thank here. vi Parallel and Serial Microfluidic Platforms for Femtosecond Laser Axotomy in Caenorhabditis elegans for Nerve Regeneration Studies Sertan Kutal Gokce, Ph.D. The University of Texas at Austin, 2016 Supervisor: Adela Ben-Yakar Understanding the molecular basis of nerve regeneration can potentiate the development of novel and efficient treatments for neurodegenerative diseases. Severing axons in the small nematode Caenorhabditis elegans (C. elegans) with femtosecond laser surgery and then observing the subsequent axonal regrowth is a promising approach to understand the molecular mechanisms of nerve regeneration in vivo. Effective and reversible immobilization of the nematodes is necessary for both axotomy and follow-up imaging. However, conventional worm handling techniques such as using anesthetics or polystyrene beads are labor-intensive and time-consuming processes, hindering high- throughput. Microfluidic devices enable the manipulation of the nematodes on a single chip with unprecedented throughput and integrity. This dissertation introduces two comprehensive microfluidic systems for femtosecond laser axotomy in C. elegans, offering several advantages over conventional techniques in terms of speed and the ability for automation of the tedious axotomy experiments. The first microfluidic system is an automated serial microfluidic platform for vii performing femtosecond laser axotomy in C. elegans. The microfluidic platform along with a custom-developed automation program isolates a single nematode from a pre-loaded population, immobilizes the nematode, and performs femtosecond laser axotomy. The full automation of the axotomy process is achieved by combining efficient image analysis methodologies with synchronized valve and flow progression in the microfluidic chip to perform multiple surgeries in a serial and automated manner. The serial automated microfluidic platform reduces the time required to perform axotomies within individual worms to ~ 17 s/worm. The second microfluidic system is a parallelized multitrap microfluidic platform, “worm hospital”, that allows on-chip axotomy, post-surgery housing for recovery, and imaging of nerve regeneration on a single chip. The microfluidic platform features 20 trapping channels for laser axotomy and subsequent post-surgery imaging, and a perfusion area to house the worms after laser axotomy. This microfluidic platform is a single-flow Polydimethylsiloxane (PDMS) layer device and has no active control PDMS layer, which reduces the fabrication and operation complexity of the chip, especially for non-expert users. The roles of neurodevelopmental genes in the Wnt/Frizzled pathway on the nerve regeneration was investigated using the “worm hospital”. In summary, the microfluidic platforms presented in this dissertation enabled performing femtosecond laser axotomy in C. elegans in a fast and repeatable manner with a controllable microenvironment. Both microfluidic platforms offer promising methodologies for prospective large-scale screening of genes involved in nerve regeneration with a high throughput in an automated manner. viii Table of Contents List of Tables ........................................................................................................ xii List of Figures ...................................................................................................... xiii Chapter 1: Introduction ............................................................................................1 1.1 Dissertation overview and objectives .......................................................2 Chapter 2: Background ............................................................................................4 2.1. C. elegans as a model organism:..............................................................4 2.2 Femtosecond laser ablation .......................................................................5 2.3 Femtosecond laser axotomy in C. elegans ................................................7 2.4 Microfluidics Overview ............................................................................8 2.5 Microfluidic devices for C. elegans research .........................................11 Chapter 3: The Fully Automated Serial Femtosecond Laser Axotomy Chip ........15 3.1 Introduction .............................................................................................15 3.2 System Overview ....................................................................................17 3.3 Microfluidic Device Design ....................................................................19 3.4 Progression of valve actuation, flow, and worm manipulation processing25 3.5 Image processing methodology for automated identification of neurons and targeting for laser axotomy ..................................................................28 3.5.1 Step 1: Identification of the worm location ................................31 3.5.2 Step 2: Detection of a neuronal cell body in the small FOV ......33 3.5.3 Step 3: Verification of neuron of interest ...................................35 3.5.4 Step 4: Laser axotomy ................................................................38 3.6 Characterization of the automated platform ...........................................39 3.6.1 Effect of chip manipulation on worm survivability ....................39 3.6.2 Timing and axotomy success rates .............................................40 3.6.3 Automated on-chip surgery axonal reconnection rates ...............44 3.7 Experimental Procedures ........................................................................46 3.7.1 Device fabrication methods ........................................................46