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FLIM) to Study Metabolism and Oxidative Stress in Biological Systems

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FLIM) to Study Metabolism and Oxidative Stress in Biological Systems UNIVERSITY OF CALIFORNIA, IRVINE Label-free fluorescence lifetime imaging microscopy (FLIM) to study metabolism and oxidative stress in biological systems DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Engineering by Rupsa Datta Dissertation Committee: Professor Enrico Gratton, Chair Associate Professor Eric O. Potma Assistant Professor Michelle A. Digman 2016 Portions of Chapter 3 © 2015 Nature Publishing Group Portions of Chapter 4 © 2016 Optical Society of America Portions of Chapter 5 © 2016 Nature Publishing Group All other material © 2016Rupsa Datta DEDICATION To my parents ii TABLE OF CONTENTS List of Figures ..................................................................................................................................................... viii List of Tables .......................................................................................................................................................... xi Acknowledgements ............................................................................................................................................ xii Curriculum Vitae ............................................................................................................................................... xiii Abstract of the Dissertation ......................................................................................................................... xvii Chapter 1 Introduction ....................................................................................................................................... 1 1.1 Metabolism and oxidative stress ........................................................................................................ 1 1.2 Techniques to study metabolism and oxidative stress .............................................................. 4 1.3 Label free fluorescence imaging .......................................................................................................... 7 1.4 NADH, a biomarker for metabolic imaging ..................................................................................... 9 1.5 Chapter Summary .................................................................................................................................. 13 Chapter 2 Methods ............................................................................................................................................ 14 2.1 Fluorescence lifetime and its measurement................................................................................ 14 2.2 Phasor Approach to Fluorescence Lifetime Imaging Microscopy (FLIM) ....................... 19 2.2.1 Advantage of the phasor analysis ............................................................................................ 19 2.2.2 The phasor transformation........................................................................................................ 21 2.3 NADH FLIM phasor ............................................................................................................................... 24 2.4 FLIM map representation ................................................................................................................... 25 2.5 Spectral phasor analysis in brief ...................................................................................................... 27 2.6 FLIM instrumentation .......................................................................................................................... 28 2.7 Chapter Summary .................................................................................................................................. 30 Chapter 3 A new approach to label-free detection of oxidative stress ......................................... 31 3.1 Introduction ............................................................................................................................................. 31 3.2 Materials and methods ........................................................................................................................ 35 3.2.1 FLIM and THG instrument ......................................................................................................... 35 3.2.2 CARS instrument ............................................................................................................................ 36 3.2.3 Raman spectroscopy .................................................................................................................... 36 3.2.4 Adipose tissue ................................................................................................................................. 37 3.2.5 HeLa cell culture and oleic acid treatment .......................................................................... 37 3.2.6 Solution preparation .................................................................................................................... 38 iii 3.2.7 Melanoma cells ............................................................................................................................... 38 3.2.8 Breast cancer cells ......................................................................................................................... 38 3.3 Results ........................................................................................................................................................ 39 3.3.1 Identification of unique a long lifetime species in white adipose tissue by FLIM 39 3.3.2 FLIM phasor signature of long lifetime species ................................................................. 44 3.3.3 Long lifetime species in brown adipose tissue .................................................................. 44 3.3.4 Long lifetime species in HeLa cells treated with oleic acid ........................................... 46 3.3.5 Use of label-free techniques to determine the origin of the autofluorescence signal ............................................................................................................................................................. 49 3.3.6 Chemical analysis by Raman spectroscopy ......................................................................... 51 3.4 LLS as biomarker for oxidative stress in cancer ........................................................................ 52 3.4.1 Melanoma ......................................................................................................................................... 52 3.4.2 Breast cancer ................................................................................................................................... 54 3.5 Discussion ................................................................................................................................................. 58 3.6 Chapter summary .................................................................................................................................. 64 Chapter 4 Metabolism and oxidative stress in hiPS cardiomyocytes ............................................ 65 Metabolism and oxidative stress in hiPS cardiomyocytes ................................................................. 65 4.1 Introduction ............................................................................................................................................. 65 4.2 Materials and methods ........................................................................................................................ 69 4.2.1 Instrumentation ............................................................................................................................. 69 4.2.2 hiPS-CM differentiation and culture....................................................................................... 70 4.2.3 Cyanide treatment ......................................................................................................................... 71 4.2.4 Hypoxia exposure .......................................................................................................................... 71 4.2.5 Drug treatments ............................................................................................................................. 71 4.2.6 In vivo staining ............................................................................................................................... 72 4.2.7 Data analysis .................................................................................................................................... 72 4.3 Results ........................................................................................................................................................ 72 4.3.1 Detection of metabolic shift in response to cyanide ........................................................ 72 4.3.2 FLIM detects metabolic response to hypoxia ..................................................................... 75 4.3.3 LLS produced in hypoxia indicating oxidative stress ...................................................... 76 4.3.4 Cardiotoxic drugs produce LLS ................................................................................................ 79 4.4 Discussion ................................................................................................................................................. 80 4.5 Chapter Summary .................................................................................................................................
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