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Multiplexed Photometry and Fluorimetry Using Multiple Frequency Channels Khaled M

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Multiplexed Photometry and Fluorimetry Using Multiple Frequency Channels Khaled M Wayne State University Wayne State University Dissertations 1-2-2013 Multiplexed Photometry And Fluorimetry Using Multiple Frequency Channels Khaled M. Dadesh Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Electrical and Computer Engineering Commons Recommended Citation Dadesh, Khaled M., "Multiplexed Photometry And Fluorimetry Using Multiple Frequency Channels" (2013). Wayne State University Dissertations. Paper 757. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. MULTIPLEXED PHOTOMETRY AND FLUORIMETRY USING MULTIPLE FREQUENCY CHANNELS by KHALED M. DADESH DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2013 MAJOR: ELECTRICAL ENGINEERING Approved by: ________________________________ Advisor Date ——————————————————— ——————————————————— ——————————————————— © COPYRIGHT BY KHALED M. DADESH 2013 All Rights Reserved DEDICATION I dedicate my humble work to the soul of my father who encouraged and supported me to be in the right path, my mother who raised me and still prays for me to be a successful person to people and community, my wife and kids, and all my family members and friends who gave me support and help to finish my dissertation. ii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. Amar Basu, who contributed tremendous time and valuable support to my research. I also appreciate Dr. Yang Zhao, Dr. Mark Ming-Cheng, and Dr. Jessica Back for their constructive comments and precious suggestions and support. I extend my appreciation to our group members in the MB laboratory. Lastly, I acknowledge all the ECE-department’s faculty and staff and the machine- shop’s technical staff for help and support. iii TABLE OF CONTENTS Dedication .......................................................................................................................... ii Acknowledgements .......................................................................................................... iii List of Figures .................................................................................................................. vii List of Tables .................................................................................................................. xvi Abbreviations ................................................................................................................ xvii CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1 Problem Statement and Motivation ........................................................................... 1 1.2 Research Objectives and Contributions ..................................................................... 3 CHAPTER 2: BACKGROUND PRINCIPLES AND LITERATURE REVIEW ...... 5 2.1 Background Principles ............................................................................................... 5 2.1.1 FDM Principle .................................................................................................. 5 2.1.3 Avalanche Photo diode (APD) ......................................................................... 6 2.1.4 Photomultiplier Tubes (PMT) .......................................................................... 6 2.1.5 Lock-in/Phase Sensitive Detection ................................................................... 7 2.1.6 Lock-in Detection in Spectrophotometry ......................................................... 9 2.1.7 Multiplexed Lock-in Detection ...................................................................... 10 2.1.8 Laser Light Modulation and Heterodyne Detection Principles ...................... 11 2.1.9 Noise and Bandwidth Analysis ...................................................................... 15 2.1.10 Multi-Color Flow Cytometry ......................................................................... 18 2.1.11 Microfluidic Chip Preparation ....................................................................... 23 2.1.12 Droplet Generation ......................................................................................... 28 iv 2.1.13 Flow Focusing ................................................................................................ 31 2.1.14 Literature Review of Existing Multiplexing Techniques ............................... 34 CHAPTER 3: CONTROL CIRCUIT DESIGN AND EMPLEMENTATION ......... 39 3.1 Photometry-FDM System ........................................................................................ 39 3.1.1 Control Circuit of Photometry-FDM System ................................................. 39 3.1.2 PCB Layout .................................................................................................... 42 3.2 Fluorimetry-FDM System ........................................................................................ 43 3.2.1 Control Circuit of Fluorimetry-FDM System ................................................ 43 3.2.1.1 Power Supply ................................................................................................. 47 3.2.1.2 Laser Driver Circuit ................................................................................... 50 3.2.1.3 Analog Multiplier Circuit .......................................................................... 52 3.2.1.4 Wide-Band Amplifier Circuit .................................................................... 54 3.2.1.5 Instrumentation Amplifier Circuit ............................................................. 55 3.2.1.6 Square Wave Voltage Controlled Oscillator ............................................. 56 3.2.2 PCB Layout .................................................................................................... 57 CHAPTER 4: PHOTOMETRY EXPERIMENTAL RESULTS ............................... 60 4. Absorbance Photometry ............................................................................................... 60 4.1.1 FDM Absorbance System Design and Operation .......................................... 60 4.1.2 Results and Discussion of the Photometry-FDM system ............................... 62 4.1.2.1 Time and Frequency Domain Measurements ............................................ 62 4.1.2.1 Multispectral Absorbance Photometry of Organic Dyes ............................. 66 4.1.2.2 Time Analysis of Droplet Microreactors ................................................... 68 v CHAPTER 5: FLUORIMETRY EXPERIMENTAL RESULTS .............................. 71 5.1 Low Speed Laser Induced Fluorescence FDM System (FDM-LIF) ....................... 71 5.1.1 System Concept and Experimental Setup ...................................................... 71 5.1.2 Results and Discussion ................................................................................... 73 5.2 A 40 MHz Frequency Multiplexed Electronic System for Multicolor Droplet Flow Cytometry ................................................................................................................ 76 5.2.1 System Concept and Experimental Setup ...................................................... 76 5.2.2 Results and Discussion ................................................................................... 80 5.2.2.1 Distinguishing Alternating Droplets .......................................................... 80 5.2.2.2 Detection Speed Limit ............................................................................... 82 5.2.2.3 Limit of Detection (LOD), Sensitivity and Cross talk ............................... 83 CHAPTER 6: APPLICATION TO CELL CYTOMETRY........................................ 87 6.1 Old Imunocytometry System ................................................................................... 87 6.2 FDM-LIF System Installation in the Old Imunocytometry System ........................ 90 6.3 Experimental Results ............................................................................................... 97 CHAPTER 7: CONCLUSIONS .................................................................................. 100 References ...................................................................................................................... 103 Abstract .......................................................................................................................... 111 Autobiographical Statement ........................................................................................ 115 vi LIST OF FIGURES Figure 1: Frequency domain of a FDM signal. ................................................................... 5 Figure 2: Photocathode radiant sensitivity of reflection mode PMTs that are made by Hamamatsu-Photonics Company. The PMT we use is from the 562U category.……………………………………………………...……..…………7 Figure 3: Lock in amplifier concept applied for a square wave sample signal (A) and another square wave reference signal (electrical not optical) (B). (C) is the product of the two signals. (D) is the lock in output after integration. Note. Signal A has a DC component, but B does not. .............................................. 10 Figure 4: Transimpedance amplifier noise model. Three noise sources are current sources and one is a voltage source ............................................................................
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