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Fluorescence Imaging of Cellular Organelles and Membrane Dynamics Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Fluorescence imaging of cellular organelles and membrane dynamics Chamari Wijesooriya Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Analytical Chemistry Commons Recommended Citation Wijesooriya, Chamari, "Fluorescence imaging of cellular organelles and membrane dynamics" (2019). Graduate Theses and Dissertations. 17807. https://lib.dr.iastate.edu/etd/17807 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Fluorescence imaging of cellular organelles and membrane dynamics by Chamari S. Wijesooriya A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Analytical Chemistry Program of Study Committee: Emily A. Smith, Major Professor Arthur Winter Young-Jin Lee Robbyn K. Anand Brett VanVeller The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Chamari S. Wijesooriya, 2019. All rights reserved. ii DEDICATION This dissertation is dedicated to my parents, husband Nuwan, loving daughter Sandali, and especially to my uncle Bimal De Silva who encouraged me to pursue my higher studies in the United States. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v ABSTRACT ................................................................................................................................... vi DISSERTATION OVERVIEW................................................................................................... viii CHAPTER 1. GENERAL INTRODUCTION ................................................................................1 OPTICAL IMAGING OF THE NANOSCALE STRUCTURE AND DYNAMICS OF BIOLOGICAL MEMBRANES ................................................................................................ 1 1.1 Introduction ......................................................................................................................... 1 1.2 Fluorescence Microscopy Techniques ................................................................................. 3 1.2.1 Single Molecule Localization Microscopy (SMLM) .................................................. 4 1.2.2 Super-Resolution Optical Fluctuation Imaging (SOFI) ............................................ 12 1.2.3 Stimulated Emission Depletion Microscopy (STED) ............................................... 14 1.2.4 Super-Resolution Structured Illumination Microscopy (SR-SIM) ........................... 18 1.2.5 Fluorescent Probes for Membrane Studies ................................................................ 19 1.3 Raman Imaging .................................................................................................................. 22 1.3.1 Surface-Enhanced Raman Spectroscopy (SERS)...................................................... 23 1.3.2 Tip-Enhanced Raman Spectroscopy (TERS) ............................................................ 25 1.3.3 Raman-Based Techniques being Developed for Membrane Studies ........................ 27 1.4. Summary and Outlook ...................................................................................................... 29 1.5 References ......................................................................................................................... 31 1.6 Figures ............................................................................................................................... 43 CHAPTER 2. COUMARIN-BASED FLUORESCENT PROBES FOR SELECTIVELY TARGETING AND IMAGING THE ENDOPLASMIC RETICULUM IN MAMMALIAN CELLS ...........................................................................................................................................54 2.1 Abstract .............................................................................................................................. 54 2.2 Introduction ....................................................................................................................... 55 2.3 Materials and Methods ...................................................................................................... 57 2.3.1 Synthesis .................................................................................................................... 57 2.3.2 Characterization of the Optical Properties of Compound 1a and 1b ......................... 58 2.3.3 Cell Culture ............................................................................................................... 59 2.3.4 Cell Viability Assay .................................................................................................. 60 2.3.5 Preparation of Cells for Microscopy Studies ............................................................ 60 2.3.6 Microscopy Experiments ........................................................................................... 61 2.3.7 Co-localization Experiment for Organelle-Specific Fluorophores ........................... 61 2.4 Results and Discussion ...................................................................................................... 62 2.4.1 Synthesis and Characterization ................................................................................. 62 2.4.2 Cell Viability Assay .................................................................................................. 63 2.4.3 Microscopy Experiments Showing Localization to the ER ...................................... 63 2.4.4 Effects of Solvent Polarity on Optical Properties ..................................................... 65 2.5 Conclusion ......................................................................................................................... 66 iv 2.6 References ......................................................................................................................... 67 2.7 Figures ............................................................................................................................... 70 2.8 Supplementary Information ............................................................................................... 74 2.8.1 Characterization of Compound 1a and 1b ................................................................. 74 2.8.2 Trypan Blue Assay .................................................................................................... 75 2.8.3 Supplementary Figures .............................................................................................. 76 CHAPTER 3. A PHOTOACTIVATABLE BODIPY PROBE FOR LOCALIZATION- BASED SUPER-RESOLUTION CELLULAR IMAGING ..........................................................85 3.1 Abstract .............................................................................................................................. 85 3.2 Introduction ....................................................................................................................... 86 3.3 Results and Discussion ...................................................................................................... 87 3.4 Conclusion ......................................................................................................................... 92 3.5 References ......................................................................................................................... 93 3.6 Tables and Figures ............................................................................................................. 97 3.7 Supplementary Information ............................................................................................. 100 3.7.1 Probe Synthesis ....................................................................................................... 100 3.7.2 Fluorescence and Photoactivation Quantum Yield Measurements ......................... 106 3.7.3 Single Molecule Localization Microscopy .............................................................. 110 3.7.4 Additional Figures ................................................................................................... 115 3.7.5 References for Supplementary Information ............................................................ 119 CHAPTER 4. EFFECT OF AMYLOID BETA 1-40 AND 1-42 PEPTIDES ON THE LATERAL DIFFUSION AND SIGNALING OF RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS (RAGE)..................................................................................121 4.1 Introduction ..................................................................................................................... 121 4.2 Materials and Methods .................................................................................................... 123 4.2.1 Cell Culture ............................................................................................................
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