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Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy Faisal Abedin Abedin University of Texas at El Paso, [email protected]

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Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy Faisal Abedin Abedin University of Texas at El Paso, Faisalabedin0@Gmail.Com University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2016-01-01 Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy Faisal Abedin Abedin University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Physics Commons Recommended Citation Abedin, Faisal Abedin, "Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy" (2016). Open Access Theses & Dissertations. 787. https://digitalcommons.utep.edu/open_etd/787 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]. STUDYING GRAZING BEHAVIOR OF CAFETERIA ROENBERGENSIS WITH TWO-PHOTON MICROSCOPY FAISAL ABEDIN Master’s Program in Physics Approved Chunqiang Li, Ph.D., Chair Cristian Botez, Ph.D. Chuan Xiao, Ph.D. Charles Ambler, Ph.D. Dean of the Graduate School Copyright © By Faisal Abedin 2016 STUDYING GRAZING BEHAVIOR OF CAFETERIA ROENBERGENSIS WITH TWO-PHOTON MICROSCOPY Faisal Abedin 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 2016 Acknowledgement At first, I would like to express my gratitude and thanks to my supervisor Dr. Chunqiang Li, Assistant Professor, Department of Physics, University of Texas at El Paso. I am truly grateful to him, not only as a supervisor but also as a mentor. His constant guidance, valuable suggestions, encouragement, innovative ideas and new experimental techniques were the driving force behind the success of the research work. He has taught me the art of quality research and independent thinking. I thank him for everything he has done for me and helping me to complete this work. It is my great pleasure to express my gratitude to Dr. Chuan Xiao, Assistant Professor, Department of Chemistry, University of Texas at El Paso, not only for providing us the samples, but also because of offering much advice during our weekly meetings. I would like to thank him for his suggestions, and continuous encouragement in my research. I would like to express my thanks to Dr. Cristian Botez, Chair , Department of Physics, University of Texas at El Paso for allowing me to use the facilities of Physics Department and for being a member of my thesis committee .Also I am grateful to him for his support and help in Physics department . Finally, I would like to express my gratitude to my parents and sisters, who raised me and trusted me with their endless love. iv Abstract Major innovations in recent years have largely revolutionized the fluorescence imaging. Two- photon fluorescence microscopy is one of them. Two photon fluorescence microscopy has evolved as an alternative to conventional single-photon confocal microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly scattering samples, and reduced phototoxicity, enabling long term imaging of photosensitive biological specimens. The inherent three-dimensional resolution of TPE microscopy has been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and submicrometer scale within thick biological specimens proved critical. In this study, we used our two-photon microscope to observe grazing behavior of fast moving marine organism Cafeteria roenbergensis with the interaction of bacteria. v Table of Content 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 1.3.1 Intensity Square Dependence versus Power and Optical Sectioning………….…………3 1.3.2 Cross-section and Colocalization……………………………………………...…………8 1.4 Experimental Setup…………………………………………………………………….……10 1.4.1 Light Source…………………………………………………………….……………….10 1.4.2 Waveplate and Polarizer ………………………………………………..………………10 1.4.3 Scanning Platform………………………………………………………...……………..13 1.4.4 Dichroic Mirror and Filters……………………………………………………….……..14 1.4.5 Objective Lens…………………………………………………………………………..15 1.4.6 Photomultiplier Tube (PMT)…………………………………………………………….16 1.4.7 3D Stage……………………………………………………………………………...…..17 1.5 Advantages of Two-Photon Microscopy………………………………………………….....18 Chapter 2: Grazing Behavior of Cafeteria roenbergensis ………………………………………21 2.1 Introduction………………………………………………………………………..…………21 2.2 Cafeteria Roenbergensis…………………………………………………………..…………21 2.3 Bacteria………………………………………………………………………………………24 2.4 Microsphere ………………………………………………………………………...……….25 2.5 Samples Preparation…………………………………………………………….……………26 vi 2.5.1 Cafeteria roenbergensis Preparation………………………………………………….……26 2.5.2 Bacteria Preparation…………………………………………………………………….….27 2.5.3 Microsphere Preparation………………………………………………………………..….28 2.6 Two-Photon Fluorescence Microscopy Imaging…………………………………………….28 2.6.1 Cafeteria roenbergensis (Cro) Imaging…………………………………………………….28 2.6.2 Bacteria Imaging……………………………………………………………………...……30 2.6.3 Carboxylate-modified FluoSphere beads Imaging…………………………………..…….32 2.6.4 Imaging for Interaction between Cafeteria roenbergensis and Bacteria …………….……32 2.6.5 Imaging for Interaction between Cafeteria roenbergensis and Microsphere………………37 2.7 Statistical Analysis of Interactions …………………………………………………….…….40 2.8 Conclusion ………………………………………………………………………..…………51 Reference ………………………………………………………………………………..………52 Vita ………………………………………………………………………………………………55 vii List of Tables 1.1 Lines per image corresponding to each frame rate…………………………………………..13 2.1 Counting number of particles (Interaction of Cro and bacteria OD 1.2) by using ImageJ software ……………………………………………………………………………..…………..42 2.2 Counting number of particles (Interaction of Cro and bacteria OD 0.6) by using ImageJ software ……………………………………………………………………………...…………..43 2.3 Counting number of particles (Interaction of Cro and bacteria OD 0.3) by using ImageJ software ……………………………………………………………………………………...…..44 2.4 Counting number of particles (Interaction of Cro and microsphere) by using ImageJ software ……………………………………………………………………………………..……………..49 viii List of Figures 1.1 Simplified scheme of the energy transition occurring under TPE regime………….…………5 1.2 Schematic of the Two-Photon Laser Scanning Fluorescence Microscope developed in the Biophotonics Laboratory of the Physics Department at UTEP……………………..……….12 1.3 One Photon Vs Two Photon Fluorescence Imaging …………………………………...……18 1.4 Comparison of Confocal Microscopy and Two-Photon Microscopy………………………..19 2.1 Cafeteria roenbergensis ……………………………………………………………………..23 2.2 Scanning electron mictoscope of Eschericha Coli (E.coli) …………………………………24 2.3 FluoSpheres Carboxylate-Modified Microscopes………………………..………………….26 2.4 Cafeteria roenbergenesis in NADH Autofluorescence ……………………………………..29 2.5 Cafeteria roenbergenesis in NADH Autofluorescence (after changing the color)…………..30 2.6 Bacteria (OD 1.2) signal with stained by SYBR Gold Stain ……………………………….31 2.7 Bacteria (OD 0.6) signal with stained by SYBR Gold Stain …………………….………….31 2.8 Bacteria (OD 0.3) signal with stained by SYBR Gold Stain …………………….………….31 2.9 Carboxylate-modified FluoSpheres beads in Red Channel………………………….………32 2.10 Carboxylate-modified FluoSpheres beads in Red Channel (after changing the color)……………………………………………………………………………………….……32 2.11 Interaction between Cro and Bacteria in different time ……………………….………….34 2.12 Interaction between Cro and Bacteria in different time…………………………………...36 2.13 Interaction between Cro and Microsphere in different time………………………..……..38 2.14 Interaction between Cro and Microsphere in different time……………………….………39 2.15 Plot of percentage of overlapping particles (between cro and bacteria) with time (in middle surface)…………………………………………………………………………………….……..45 ix 2.16 Plot of percentage of overlapping particles (between cro and bacteria) with time (for 3 different experiments)……………………………………………………………………………46 2.17 Plot of percentage of overlapping particles (between cro and bacteria) with time (in top surface)……………………………………………………………………………..…………….47 2.18 Plot of percentage of overlapping particles (between cro and bacteria) with time (in both surfaces)…………………………………………………………………………………....…….48 2.19 Plot of percentage of overlapping particles (between cro and microsphere) with time (in both surfaces)…………………………………………………………………………….………50 x Chapter 1: Two- photon Microscopy (TPM) 1.1 Introduction In light of non-linear optical wonders different laser-scanning techniques have been made in the latest a quarter century. Two-photon excited fluorescence microscopy (TPM), Second Harmonic Generation (SHG) microscopy and Coherent Anti stokes Raman Scattering (CARS) microscopy are a portion of the intense imaging devices. The upsides of these creative system have empowered incredible advancements in biological imaging, particularly in thick tissue and live creature studies than the ordinary optical microscopy strategies. A standout amongst the most essential late innovations in biological imaging is two-photon fluorescence microscopy (TPM). This innovation empowers noninvasive investigation of organic specimens in three dimensions with submicrometer resolution. Two-photon fluorescence
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