Wide-field Structured Illumination Microscopy for Fluorescence and Pump-Probe Imaging By Yang-Hyo Kim B.S., Mechanical and Aerospace Engineering Seoul National University (2005) S.M., Mechanical Engineering Massachusetts Institute of Technology (2007) Submitted to the Department of Mechanical Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2019 C 2018 Massachusetts Institute of Technology. All rights reserved Signature redacted Signature of Author: Department of Mechanical Engineering Signature redactedIan i5, 2019 Certified by: Peter T. C. So Professor of Mechanical Engineering and Engineering .s ervisor Signature redacted Accepted by: MASSACHUSETS INS1TTWE Nic las Hadjiconstantmou OF TECHNOLOGY Chairman of the Departmental Committee for Graduate Students IEB 25 2019 LIBRARIES ARCHIVES Wide-field Structured Illumination Microscopy for Fluorescence and Pump-Probe Imaging By Yang-Hyo Kim Submitted to the Department of Mechanical Engineering on Jan 15, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Mechanical Engineering Abstract The optical resolution of microscopy is limited by the wave-like characteristic of the light. There are many recent advances in overcoming this diffraction limited resolution, but mostly focused on fluorescent imaging. Furthermore, there are few non-fluorescence wide-field super-resolution techniques that can fully utilize the applicable laser power to optimize imaging speed. Structured illumination microscopy is a super-resolution method that relies on patterned excitation. This thesis has presented novel applications of structured illumination microscopy to surface plasmon resonance fluorescence and pump-probe scattering imaging. First, structured illumination microscopy was introduced to surface plasmon resonance fluorescence imaging for high signal-to- noise and high resolution. Secondly, a theoretical framework for three-dimensional wide-field pump-probe structured illumination microscopy has been developed to increase the lateral resolution and enable depth sectioning. Further, structured illumination wide-field photothermal digital phase microscopy is proposed as a high throughput, high sensitivity super-resolution imaging tool to diagnose ovarian cancer. Finally, I have derived the exact analytical solution to the heat conduction problem in which a sphere absorbs temporally modulated laser beam for photothermal microscopy. The proposed method also has a great potential to be applied to other pump-probe modalities such as transient absorption and stimulated Raman scattering. Thesis Supervisor: Peter T. C. So Title: Professor of Mechanical Engineering and Biological Engineering 2 Acknowledgement First, I would like to thank my thesis advisor, Prof. Peter So for his generosity and guidance. His door was always open and I could ask whatever silly question to him. Whenever discussion needed, I could talk fact-to-face physically or through Skype meeting even when he was waiting for the airplane in the international airport. I also would like to thank Prof. Shyamsunder Erramilli and Prof. Mi Hong for their advice and help. They were another greatest support for my study and life at MIT. I would like to express my gratitude to Prof. George Barbastathis and Prof. Nicholas Xuanlai Fang for their advice, guidance and generous support as my committee members. I appreciate Prof. Colin J. R. Sheppard, Prof. K. C. Toussaint, Jr., Prof. Conor L. Evans, and Prof. Aydogan Ozcan for their academic and personal support. I also thank all the former and current So Lab members and LBRC members, especially Daekeun Kim, Hyuksang Kwon, Euihean Chung, Maxine Jonas, Heejin Choi, Jae Won Cha, Dimitris Tzeranis, Hayden Huang, Ki Hean Kim, Christopher Rowlands, Elijah Yew, Vijay Raj Singh, Yun-Ho Jang, Jong Kang Park, Barry Masters, Yi Xue, Baoliang Ge, Dushan Wadduwage, Murat Yildirim, Sossy Megerdichian, Zahid Yaqoob, Jeong Woong Kang, Sungsam Kang, Surya Pratap Singh, Renjie Zhou, Luis H. Galindo, and Christine Brooks for their academic advice, numerous help, and warm-hearted support. All of my collaborators, Wai Teng Tang and Sinyoung Jeong, sincerely worked together with me and their positive energy always encouraged me to move forward. I want to thank all the people who have been with me in my Boston life including Korean Graduate Student Association members in Mechanical Engineering. The administrators of Mechanical Engineering and Biological Engineering, Leslie Regan and Olga Parkin, took care of various issues I generated with magically smooth and timely manner. I cannot imagine how I can express my appreciation to my family: Hong Sik Kim, Gi Soon Lee, Mi Yeon Kim, Hee Chang Jeong, Young Hee Kim, Kyung Suk Jeong, and Buhyun Yun. Especially I thank my wife, Hyung Mee Jeong for her constant support, sacrifice and endless love. I would like to acknowledge my funding sources and sponsors: National Institutes of Health (9P41EB015871-26A1, 5R01NS051320, 4R44EB012415, and 1R01HL121386-OlAl), National Science Foundation (CBET-09395 11), Hamamatsu Corporation, Singapore-Massachusetts Institute of Technology Alliance for Research and Technology (SMART) Center, BioSystems and Micromechanics (BioSyM), and Samsung Scholarship. 3 Table of Contents 1. Introduction ................................................................................................................................. 10 1.1. M icroscopic image formation......................................................................................... 10 1.2. Super-resolution m icroscopy ........................................................................................... 11 1.3. Imaging speed improvem ent in m icroscopy ................................................................... 14 1.4. 3D wide-field m icroscopy technologies ......................................................................... 15 1.5. Review of super-resolution m icroscopy.............................................................................. 17. 1.6. Objectives ........................................................................................................................... 17 2. Wide-field extended-resolution fluorescence microscopy with standing surface-plasmon- resonance waves............................................................................................................................... 20 2.1. Introduction......................................................................................................................... 20 2.2. Principle of SW -SPRF m icroscopy................................................................................. 22 2.2.1. Fluorescence excitation in SW -SPRF microscopy .............................................. 22 2.2.2. Fluorescence detection in SW -SPRF m icroscopy................................................. 24 2.2.3. Im age formation in SW -SPRF microscopy ......................................................... 27 2.3. M ethods and Experimental Setup .................................................................................. 30 2.4. Signal intensity comparison of different im aging modes................................................. 31 2.5. Calculation of the transmitted intensity and collected emission photons ........................ 33 2.6. Point spread function measurement of SW-SPRF microscope ........................................ 34 2.7. Conclusion .......................................................................................................................... 36 3. Theoretical framework for three-dimensional wide-field pump-probe structured illum ination m icroscopy ................................................................................................................. 39 3.1. Introduction......................................................................................................................... 39 3.2. Principle of 3D SIM pump-probe m icroscopy.................................................................... 41 3.2.1. Configuration of a wide-field pump-probe structured illumination microscope ...... 41 3.2.2. Theoretical fram ework......................................................................................... 43 3.2.2.1. 3D CTF and getting a 3D object information with a 2D detector .............. 43 3.2.2.2. Principle of 3D wide-field pump-probe structured illumination microscope. 48 3.2.2.3. Ideal choice of structured illumination phase set for 3D wide-field pump- probe structured illum ination m icroscope .............................................................. 54 3.3. M ethods for num erical simulation .................................................................................. 56 4 3 .4 . R esu lts................................................................................................................................. 5 7 3.4.1. Imaging of a planar target: Calibration Chart ........................................................ 57 3.4.2. Imaging of a non-planar target: a 3D M IT Logo ................................................... 60 3.4.3. Imaging of Biomolecules in HeLa Cells............................................................... 61 3.5. Discussion........................................................................................................................... 62 3.6. Conclusion .........................................................................................................................