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Dissertation Single Molecule Fluorescence DISSERTATION SINGLE MOLECULE FLUORESCENCE MEASUREMENTS OF COMPLEX SYSTEMS Submitted by Sanaz Sadegh Department of Electrical and Computer Engineering In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2017 Doctoral Committee: Advisor: Diego Krapf Michael Tamkun Edwin Chong Ashok Prasad Copyright by Sanaz Sadegh 2017 All Rights Reserved ABSTRACT SINGLE MOLECULE FLUORESCENCE MEASUREMENTS OF COMPLEX SYSTEMS Single molecule methods are powerful tools for investigating the properties of complex systems that are generally concealed by ensemble measurements. Here we use single molecule fluorescent measurements to study two different complex systems: 1=f noise in quantum dots and diffusion of the membrane proteins in live cells. The power spectrum of quantum dot (QD) fluorescence exhibits 1=f β noise, related to the intermittency of these nanosystems. As in other systems exhibiting 1=f noise, this power spectrum is not integrable at low frequencies, which appears to imply infinite total power. We report measurements of individual QDs that address this long-standing paradox. We find that the level of 1=f β noise for QDs decays with the observation time. We show that the traditional description of the power spectrum with a single exponent is incomplete and three additional critical exponents characterize the dependence on experimental time. A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mam- malian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal. ii ACKNOWLEDGEMENTS First and foremost I want to express my sincere appreciation and gratitude to my thesis advisor Dr. Diego Krapf for the continuous support of my Ph.D study and related research, for his patience, motivation, and immense knowledge. Thank you for guiding me to grow as a research scientist. I would also like to thank the rest of my thesis committee: Prof. Michael Tamkun, Dr. Ashok Prasad, and Prof. Edwin Chong, for their insightful comments and encouragement ans support. I thank past and present members of Krapf group: Xinran Xu, Jenny Higgins, Aubrey Weigel, Bryce Schroder, Patrick Mannion and Kanti Nepal. The group has been a source of friendships as well as good advice and collaboration. I also thank Phil Fox, Liz Akin, Ben Johnson, Nasim Rad, Mahsa Ghorbani and Elaheh Alizadeh for the stimulating discussions, constant support and friendship. Great thanks are also due to Leif Anderson for building this LATEX document class, allowing me to meet the graduate school formatting requirements with no effort on my part. Last but not the least, I would like to thank my family for all their love and encourage- ment. For my parents who raised me with love and supported me in all my pursuits. For the unconditional love and support of my brother and sister. And most of all for my loving, supportive, encouraging, and patient husband Farshad whose support during my Ph.D. is so appreciated. This accomplishment would not have been possible without them. iii TABLE OF CONTENTS ABSTRACT.......................................................................... ii ACKNOWLEDGEMENTS. iii LIST OF TABLES . vii LIST OF FIGURES. viii LIST OF ABBREVIATIONS. x Chapter 1. INTRODUCTION . 1 1.1. The 1=f noise in intermittent quantum dots. 2 1.2. Single molecule measurements in the plasma membrane of the cells . 3 1.3. Overview of this dissertation . 5 Chapter 2. 1=F NOISE FOR INTERMITTENT QUANTUM DOTS EXHIBITS NONSTATIONARITY AND CRITICAL EXPONENTS. 7 2.1. Introduction . 7 2.2. Theoretical model. 9 2.3. Results . 11 2.4. Discussion . 22 2.5. Conclusions . 26 2.6. Experimental methods: quantum dot imaging . 26 Chapter 3. FLUCTUATIONS OF 1=F NOISE IN INTERMITTENT QDS . 28 3.1. Introduction . 28 3.2. Results . 29 iv 3.3. Discussion . 37 Chapter 4. THE PLASMA MEMBRANE IS COMPARTMENTALIZED BY A SELF-SIMILAR CORTICAL ACTIN MESHWORK. 39 4.1. Introduction . 39 4.2. Results . 41 4.3. Discussion . 57 4.4. Materials and methods. 60 Chapter 5. CONFINED LATERAL DIFFUSION: ROLE OF THE DISTRIBUTION OF CONFINEMENT SIZES . 68 5.1. Introduction . 68 5.2. Theoretical model. 70 5.3. One dimensional (1D) diffusion . 74 5.4. Two dimensional (2D) diffusion . 78 5.5. Discussion . 83 5.6. Methods . 83 Chapter 6. PLASMA MEMBRANE PROTEIN DYNAMICS AT THE KV2.1/ER/PM JUNCTION. 85 6.1. Introduction . 85 6.2. Results . 86 6.3. Discussion . 95 BIBLIOGRAPHY. 96 Appendix A. PROTOCOLS . 118 v A.1. TIRF microscope alignment . 118 A.2. Splitting cells. 120 A.3. Cell transfection. 121 A.4. Live cell imaging preparation . 123 A.5. Cell fixation and labeling . 124 A.6. STORM imaging . 125 Appendix B. SOFTWARE. 126 B.1. Single particle tracking using u-track . 126 B.2. MSD analysis . 128 B.3. Image registration . 131 B.4. PALM reconstruction. 136 B.5. STORM reconstruction . 137 B.6. Generating Mittag-Leffler distribution . 139 B.7. Box counting fractal.
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