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THE IMPLEMENTATION OF OPTOFLUIDIC MICROSCOPY ON A CHIP SCALE AND ITS POTENTIAL APPLICATIONS IN BIOLOGY STUDIES Thesis by Lap Man Lee In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2012 (Defended September 12th, 2011) ii © 2012 Lap Man Lee All Rights Reserved iii Acknowledgement The completion of my thesis is based on support and help from many individuals. First, I would like to express my gratitude to my PhD advisor Prof. Changhuei Yang for offering me a chance to work on an emerging research field of optofluidics and participate in the development of optofluidic microscopy (OFM), which leads to many successful results. His guidance has led the OFM project from an elegant engineering idea to reality. His enthusiasm and creativity in conducting academic research is forever young, motivating us to pursue excellence in our projects. I thank him for introducing me to biophotonics and allowing me to play a role in contributing to the field. I want to thank Prof. Yu-Chong Tai for being my thesis committee chair. His pioneering work in MEMS has always been my motivation to pursue something ‘big’ in the world of ‘small’. You will find yourself learning something new every time you interact with him, not only in science but also in life. I want to thank Prof. Chin-Lin Guo for the discussion after the committee meeting. I find his advice very useful. I am thankful for suggestions from Prof. Azita Emami, which made my PhD work more complete. I also acknowledge my candidacy committee members, Prof. Yu-Chong Tai, Prof. Mory Gharib and Prof. Chin-Lin Guo for passing me. Dr. Xin Heng, Dr. Xiquan Cui, Sean Pang, Seung Ah Lee, and Chao Han are my teammates in OFM. We work closely together and exchange research experience, and this greatly accelerates the progress of our OFM projects. Dr. Jigang Wu was my mentor and labmate in the last two years of my PhD study. He is always determined to solve complicated research problems in a direct, prompt, and smart way. I also want to thank Dr. Meng Cui, Jian Ren, Guoan Zheng, Zheng Li, Dr. Zahid Yaqoob, and Dr. Benjamin Judkewitz for teaching me optics. I joined the group with practically zero knowledge in optics. Fortunately, they are never selfish and always patient, even when I ask very simple optics questions. I am so grateful to have Dr. Emily McDowell and Ying Min Wang as my bioengineering iv buddies in the group. They give me a lot of support when I run into problems. Anne Sullivan, Agnes Tong, Christine Garske, and Ya-Yun Liu and are our group manager and secretaries. They are very helpful to supply and run the lab safely and smoothly. I need to thank Ali Ghaffari, the lab manager of Watson Microfludic Foundary, and Bophan Chim, Guy DeRose, and Melissa Melendes from the Kavli NanoScience Institute at Caltech. They have helped me to solve many technical problems in fabrication. I am grateful to Justin Kim in Tai’s group, who helped me to coat parylene for devices. I appreciate the help and care from Linda Scott, the secretary of the bioengineering option and Icy Ma from the grad student office. I also want to thank Dr. Felicia Hunt, the Associate Dean of Graduate Studies for the discussions. Here I have to give credit to my masters and undergraduate advisors, Prof. Yitshak Zohar at the University of Arizona and Prof. Yi-Kuen Lee at Hong Kong University of Science and Technology, who brought me to the fascinating research field of microfluidics and BioMEMS, taught me fluid mechanics and helped me to define the right way to conduct experimental work. I am indebted to Prof. Sandra Troian, who allowed me to audit her beautiful fluid mechanics class at Caltech. I am lucky to have a mom and dad who give me complete freedom, not pressure, to decide what I like and want to do in my life. I cherish the love and care from my girlfriend, May Lam. I owe them so much so much. I appreciate the friendship from the Hong Kong student community at Caltech, for their support and suggestions throughout my entire journey as a PhD student. I feel grateful to my bioengineering classmates in my year, Eugene Buchko, Fiona Chandra, Josh Michener, and Hesham Azizgolshani. Thanks for their help and encouragement in the long preparation of my qualifying exam. I want to thank Chi-kwan Chan from Arizona, Lilian Wong and Heywood Tam from Caltech. They have been my fountain of wisdom on many problems. It happens they are all physics or pure math PhDs. Finally, I would like to acknowledge funding from the Croucher Foundation in Hong Kong, which arrived just at the right time and allowed me to continue my PhD study at Caltech. v Abstract This thesis presents an effort to miniaturize conventional optical microscopy to a chip level using microfluidic technology. Modern compound microscopes use a set of bulk glass lenses to form magnified images from biological objects. This limits the possibility of shrinking the size of a microscope system. The invention of micro/nanofabrication technology gives hope to engineers who want to rethink the way we build optical microscopes. This advancement can fundamentally reform the way clinicians and biologists conduct microscopy. Optofluidic microscopy (OFM) is a miniaturized optical imaging method which utilizes a microfluidic flow to deliver biological samples across a 1-D or 2-D array of sampling points defined in a microfluidic channel for optical scanning. The optical information of these sampling points is collected by a CMOS imaging sensor on the bottom of the microfluidic channel. Although the size of the OFM device is as small as a US dime, it can render high resolution images of less than 1 μm with quality comparable to that of a bulky, standard optical microscope. OFM has a good potential in various biological applications. For example, the integration of an OFM system with high-speed hydrodynamic focusing technology will allow very large scale imaging-based analysis of cells or microorganisms; the compactness and low cost nature of OFM systems can enable portable or even disposable biomedical diagnostic tools for future telemedicine and personalized health care. vi Table of Contents Acknowledgements .......................................................................................................................iii Abstract ..........................................................................................................................................v Table of Contents...........................................................................................................................vi List of Figures……………............................................................................................................ ix List of Tables…………….............................................................................................................xv Nomenclature................................................................................................................................xvi Chapter 1: Introduction ………………………………………………………………………1 1.1 Optical Microscopy……………………………………………………………………2 1.1.1 Resolution of Imaging Systems…………………………….…..…………4 1.1.2 Miniaturization of Optical Microscopy…………………….…..…………8 1.2 Full-Field versus Scanning Optical Microscopy……………………………………11 1.2.1 Confocal Microscopy (by moving the light spot)……………...………13 1.2.2 Wide Field-of-View Microscopy (by moving the sample)…………..17 1.3 Optofluidic Microscopy — A Brief Review………….……………………………18 1.4 Introduction to Microfluidics.………………………………………………………20 1.4.1 Optofluidic Integration……………….…..…………………………24 1.5 Structure of the Thesis…………...……………………………………………..……25 References……………….…………...……………………………………………..……26 Chapter 2: Implementation of Optofluidic Microscopy ………….……………...........................30 2.1 Imaging Formation…...……..………………………………………………………31 2.1.1 Optical Resolution.……………………………………………………32 2.1.2 Sampling Problem.……………………………………………………33 2.2 Microfluidics for Sample Transport………………………………………………36 2.2.1 Pressure-Driven Flow…..………………………………………………40 2.2.2 Electrokinetic-Driven Flow…..…………………………………………47 2.2.3 Gravity-Driven Flow……….....…………………………………………53 References……………….…………...……………………………………………..……55 vii Chapter 3: Collection-Mode Optofluidic Microscopy..…………….…………............................57 3.1 Single-Layer Structure……..………………………………………………………59 3.1.1 Using Apertures in OFM Imaging………………………………………60 3.1.2 Design and Fabrication...………………………………………………62 3.1.3 Demonstration in Bright Field OFM Imaging without Focusing Ability..66 3.1.3.1 Optical Resolution, Acceptance Angle, and Image Contrast…..71 3.2 Two-Layers Structure……..…………………………………………………………73 3.2.1 Using a Fresnel Zone Plate as a Light Collection Unit in OFM Imaging..76 3.2.2 Design and Fabrication Process...………………………………………81 3.2.3 Demonstration in Bright Field FZP-Aperture OFM Imaging …………88 3.2.3.1 Optical Resolution and Depth of Focus...............................……92 3.2.4 Demonstration in Dark Field FZP-Aperture OFM Imaging ……………98 References……………….…………...…………………………………………………104 Chapter 4: Illumination-Mode Optofluidic Microscopy ………….…………............................107 4.1 Holographic Approach to Create Focused Light Spots…..………………………109 4.1.1 Demonstration in Bright Field Holographic OFM Imaging……………112 4.2 FZP Approach to Create Focused Light Spots……………..….…………….……114 4.3 Talbot Approach to Create Focused Light Spots…………………………….……117 4.4 Limitations on Illumination-Mode OFM Imaging.…………………………………119 References……………….…………...…………………………………………………122