MEASURING THE MECHANICAL PROPERTIES OF PRIMARY CILIA WITH AN OPTICAL TRAP TARA DIBA Bachelor of Science in Physics Azad University, Central Tehran December, 2011 Submitted in partial fulfillment of requirements for the degree MASTER OF SCIENCE IN BIOMEDICAL ENGINEERING at the CLEVELAND STATE UNIVERSITY December, 2015 ©COPYRIGHT BY TARA DIBA 2015 ii We hereby approve this thesis for (Tara Diba) Candidate for the Master of Science in Biomedical Engineering degree for the Department of Chemical and Biomedical Engineering and the CLEVELAND STATE UNIVERSITY College of Graduate Studies _________________________________________________________________ Thesis Chairperson, (Dr. Andrew Resnick) Physics, Cleveland State University/Dec.4.2015 Department & Date _________________________________________________________________ Thesis Committee Member, (Dr. Chandra Kothapalli) Chemical and Biomedical Engineering, Cleveland State University/ Dec.4.2015 Department & Date _________________________________________________________________ Thesis Committee Member (Dr. Christopher L. Wirth) Chemical and Biomedical Engineering, Cleveland State University/ Dec.4.2015 Department & Date ________________________________________________________________ Thesis Committee Member (Dr. Moo Yeal Lee) Chemical and Biomedical Engineering, Cleveland State University/ Dec.4.2015 Department & Date Student’s Date of Defense: (Dec.4.2015) iii ACKNOWLEDGEMENTS I would like to first and foremost express my appreciation and sincere gratitude to Dr. Andrew Resnick for providing me this wonderful opportunity to conduct research under his astute guidance. His boundless energy, wonderful analytical skills, cool and calm composure, and motivational power has made this experience a truly memorable one. I am sure this will stand me in good stead in my future research and professional career as well. I sincerely thank him for introducing me to the research of biomedical optics. Because of him, I was able to work on many intriguing projects and learn more than I ever dreamed possible. I must also acknowledge the seemingly infinite support and kind advice from Dr. Chandra Kothapalli and Dr. Moo Yeal Lee as well. I also would like to thank Dr. Joanne Belovich for playing a pivotal role in my thesis. I owe my deepest thanks to my parents who have always stood by me with patience and satisfaction, they guided me through life and I wouldn’t have been able to finish my degree without them. Moreover, I offer my regards and blessing to all of the people, especially Rebecca Laird and Darlene Montgomery, who did not hesitate to help me in my entire life in the US. iv MEASURING THE MECHANICAL PROPERTIES OF A PRIMARY CILIA WITH AN OPTICAL TRAP TARA DIBA ABSTRACT Nonmotile primary cilia are slender subcellular structures that extend from the mother centrosome, are typically several microns long, and are used by eukaryotic cells to sense fluid flow. Cilia perform various roles to maintain tissue homeostasis through multiple signaling pathways, and their sensing ability is not restricted to physical stimuli but also biochemical stimuli from the extracellular environment. Cilia play a mechanosensory role in numerous tissues including kidney, liver and bone; where mechanical deflection of cilia due to mechanical loading leads to a cellular response. However, the relationship between cilia mechanical responses and downstream regulatory processes that are ciliary- initiated is still unknown. Optical tweezers provide a unique method to mechanically stimulate a primary cilium because of the noncontact nature of the method. We present a method to measure the mechanical properties of a primary cilium by exciting a resonant oscillation of the cilium. This is done by by applying an optical force directly to the cilium. We will show that the resonant frequency of cilia can be used to extract mechanical properties of the cilium base. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ....................................................................................................................... IV ABSTRACT ........................................................................................................................................... V NOMENCLATURE ................................................................................................................................. IX LIST OF TABLES .................................................................................................................................... XI LIST OF FIGURES ................................................................................................................................. XII CHAPTER I ............................................................................................................................................ 1 INTRODUCTION AND OBJECTIVE OF THE THESIS ................................................................................... 1 1.1 INTRODUCTION ........................................................................................................................... 1 1.2 MOTIVATION ............................................................................................................................... 2 1.3 OBJECTIVE ................................................................................................................................... 3 CHAPTER II ........................................................................................................................................... 4 HISTORY AND ADVANCEMENT OF OPTICAL TWEEZERS ......................................................................... 4 2.1 INITIAL DEVELOPMENT OF OPTICAL TWEEZERS .......................................................................... 4 2.2 BASIC OPTICS ............................................................................................................................... 6 CHAPTER III ........................................................................................................................................ 11 THEORY AND APPLIED FORCE ............................................................................................................. 11 3.1 THEORY OF OPTICAL TWEEZERS ................................................................................................ 11 3.2 OPTICAL FORCES ....................................................................................................................... 14 vi 3.2.1 RAYLEIGHT SCATTERING .............................................................................................. 17 3.2.2 RAY OPTICS APPROXIMATION ..................................................................................... 22 3.2.3 GENERALIZED LORENTZ MIE THEORY (r~λ) ............................................................ 24 3.3 FORCE CALIBRATION ................................................................................................................. 25 3.4 QPD POSITION DETECTION ........................................................................................................ 27 CHAPTER IV ........................................................................................................................................ 29 MOTILE AND NONMOTILE CILIA ......................................................................................................... 29 4.1 CILIA .......................................................................................................................................... 29 4.2 PRIMARY CILIA .......................................................................................................................... 32 4.3 CILIOPATHY ............................................................................................................................... 35 CHAPTER V ......................................................................................................................................... 38 DEVELOPED MODEL OF THE PRIMARY CILIUM .................................................................................... 38 5.1 CILIARY MECHANICS AND MODELS ........................................................................................... 38 CHAPTER VI ........................................................................................................................................ 41 METHODS AND MATERIALS ................................................................................................................ 41 6.1 METHODS .................................................................................................................................... 41 6.2 CELL CULTURE ........................................................................................................................... 42 6.3 IMMUNOCYTOCHEMISTRY ....................................................................................................... 43 6.4 OPTICAL TWEEZERS APPARATUS ............................................................................................... 44 6.4.1 Major Components Description ............................................................................................ 47 CHAPTER VII ....................................................................................................................................... 50 PRIMARY CILIUM TRAPPING ............................................................................................................... 50 vii 7.1 TRAPPING PROTOCOLE ............................................................................................................