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Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy Syeed Ehsan Ahmed University of Texas at El Paso, [email protected]

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Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy Syeed Ehsan Ahmed University of Texas at El Paso, Eshanahmed2012@Gmail.Com University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2017-01-01 Imaging Live Drosophila Brain With Two-Photon Fluorescence Microscopy Syeed Ehsan Ahmed University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Biophysics Commons, and the Optics Commons Recommended Citation Ahmed, Syeed Ehsan, "Imaging Live Drosophila Brain With Two-Photon Fluorescence Microscopy" (2017). Open Access Theses & Dissertations. 397. https://digitalcommons.utep.edu/open_etd/397 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. IMAGING LIVE DROSOPHILA BRAIN WITH TWO-PHOTON FLUORESCENCE MICROSCOPY SYEED EHSAN AHMED Master’s Program in Physics APPROVED: Chunqiang Li, Ph.D., Chair Cristian E. Botez, Ph.D. Chuan Xiao, Ph.D. Charles Ambler, Ph.D. Dean of the Graduate School Copyright © by Syeed Ehsan Ahmed 2017 Dedication Dedicated to my beloved family and friends. Thank you for believing in me and helping me throughout my journey. IMAGING LIVE DROSOPHILA BRAIN WITH TWO-PHOTON FLUORESCENCE MICROSCOPY by SYEED EHSAN AHMED, B.Sc. in Physics THESIS Presented to the Faculty of the Graduate School of The University of Texas at El Paso in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Physics THE UNIVERSITY OF TEXAS AT EL PASO August 2017 ACKNOWLEDGEMENTS All praise and worthiness goes to Almighty Merciful Allah who has given me the opportunity, strength and ability to complete this work. I would like to express my profound gratitude to my advisor, Dr. Chunqiang Li, for his mentorship, supervision and invaluable suggestions during my research. His continuous assessment and appropriate guidance enabled me to develop as a scientist and complete my research effectively. I would like to thank Dr. Cristian Botez for his enthusiasm and perseverance as the chair of the department of Physics and for the kindness and guidance he has showed during our interactions. I would also like to thank all my group members in the Biophotonics laboratory, especially Dr. Yu Ding for his constant support and guidance throughout my thesis work. I would like to take the opportunity to express my gratitude to all members of the faculty I have encountered from the Department of Physics at UTEP through their lectures, guidance and the support outside the classroom. And lastly, my sincerest gratitude goes to my parents and my family for their continuous support in every situation towards the completion of my thesis. v ABSTRACT Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non- radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ………………………………………………………………...V ABSTRACT ………………………………………………………………………………...Vi TABLE OF CONTENTS …………………………………………………………………...Vii LIST OF TABLES …………………………………………………………………………..iX LIST OF FIGURES ………………………………………………………………………….X CHAPTER 1: TWO-PHOTON MICROSCOPY.............................................................................1 1.1 INTRODUCTION .........................................................................................................1 1.2 HISTORY OF TWO-PHOTON MICROSCOPY .........................................................2 1.3 THEORY OF TWO-PHOTON ABSORPTION ...........................................................3 1.4 EXPERIMENTAL SETUP OF TWO-PHOTON FLUORESCENCE MICROSCOPE ............................................................................................................15 1.4.1 LIGHT SOURCE ................................................................................................15 1.4.2 WAVEPLATE AND POLARIZER ...................................................................16 1.4.3 SCANNING PLATFORM..................................................................................17 1.4.4 DICHROIC MIRRORS ......................................................................................19 1.4.5 BANDPASS FILTERS .......................................................................................20 1.4.6 OBJECTIVE LENS ............................................................................................20 1.4.7 PHOTOMULTIPLIER TUBE (PMT) ................................................................22 1.4.8 THREE DIMENSIONAL STAGE .....................................................................23 1.5 ADVANTAGES OF TWO-PHOTON MICROSCOPY .............................................23 CHAPTER 2: LIVE IMAGING OF DROSOPHILA MELANOGASTER BRAIN USING TWO-PHOTON MICROSCOPY .........................................................................................27 2.1 INTRODUCTION ..............................................................................................27 2.2 FÖRSTER RESONANCE ENERGY TRANSFER (FRET) ..............................28 vii 2.3 PRINCIPLE OF FRET .......................................................................................28 2.3.1 THE RATE OF ENERGY TRANSFER 푘푇 .......................................................34 2.3.2 ENERGY TRANSFER EFFICIENCY E ...........................................................35 2.3.3 ORIENTATIONAL FACTOR 휅2 ......................................................................36 2.3.4 SPECTRAL OVERLAP INTEGRAL ................................................................37 2.4 CYCLIC ADENOSINE MONOPHOSPHATE (CAMP) ...................................38 2.5 DOPAMINE .......................................................................................................39 2.6 FLUOROPHORES (CFP-YFP PAIRS) .............................................................40 2.7 EXCHANGE PROTEIN ACTIVATED BY CYCLIC AMP (EPAC1) .............41 2.8 TWO-PHOTON IMAGING LIVE DROSOPHILA BRAIN USING FRET FOR CAMP DYNAMICS ..................................................................................42 2.8.1 SAMPLE PREPARATION ................................................................................42 2.8.2 EXPERIMENTAL METHOD ............................................................................44 2.8.3 IMAGE PROCESSING USING IMAGEJ .........................................................46 2.8.4 RESULTS ...........................................................................................................47 2.9 CONCLUSION ...................................................................................................53 REFERENCES ..............................................................................................................................54 VITA........................................................................................................................................57 viii LIST OF TABLES Table 1.1 – Lines per image corresponding to different frame rates ……………………………18 Table 2.1 – Composition of hemolymph like saline …………………………………………….43 Table 2.2 – Composition of Dopamine, molecular weight and preparation …………………….44 ix LIST OF FIGURES Figure 1.1 - Jablonski diagram of One-Photon Excitation ………………………………………3 Figure 1.2 - Jablonski diagram of Two-Photon Excitation ………………………………………4 Figure 1.3 - Probability density distribution of Eq. (1.27) ……………………………………....13 Figure 1.4 - Diagram of intensity controlling system …………………………………………...17 Figure 1.5 –Schematic of two-photon microscope developed at UTEP ………………………...20 Figure 1.6 - Focal plane of single-photon Vs two-photon fluorescence microscopy …………...24 Figure 1.7 - Comparison between confocal microscopy and two-photon microscopy …………25 Figure 2.1 - Jablonski diagram illustrating FRET for TPE ……………………………………..26 Figure 2.2 – Energy transfer between D-A pair ………………………………………………...30 Figure 2.3 – FRET efficiency plot ………………………………………………………………36 Figure 2.4 – Emission and absorption spectra of D-A pair ……………………………………..38 Figure 2.5 – 2D skeletal formula of cAMP ……………………………………………………..39 Figure 2.6 – 2D skeletal formula of Dopamine …………………………………………………39 Figure 2.7 – (a) CFP and YFP spectral overlap with one-photon excitation (b) CFP and YFP emission for different TPE excitation wavelength ……………………………………………...41 Figure 2.8 – Cyclic AMP dependent Epac1 configuration ……………………………………42 Figure 2.9 - Schematic Illustration of In Vivo Preparation……………………………………...45 Figure 2.10 - Mushroom Bodies with Targeted Region (γ-lobe) (a) Bathed in Hemolymph like Solution and (b) Washed and again Bathed with Dopamine Solution…………………………..46 Figure 2.11 - (a) CFP (b) YFP
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