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Expansion Microscopy for Brain Mapping Expansion Microscopy for Brain Mapping The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kang, Jeong Seuk. 2019. Expansion Microscopy for Brain Mapping. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029733 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Expansion Microscopy for Brain Mapping by Jeong Seuk Kang B.S. Electrical Engineering and Computer Sciences/ Materials Science and Engineering University of California, Berkeley (2015) S.M. Applied Physics Harvard University (2017) Submitted to the Harvard John A. Paulson School of Engineering and Applied Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Applied Physics Harvard University May 2019 © 2019 Jeong Seuk Kang All rights reserved Dissertation Advisor: Professor Edward S. Boyden Jeong Seuk Kang Expansion Microscopy for Brain Mapping Abstract More than one billion people in the world suffer from brain disorders. To address this, more than one trillion US dollars are spent to develop the drugs, but ~92% fail to receive clinical approval. Among many potential reasons why treating brain disorders has been strikingly difficult, one reason could be that the complexity of neural circuitry and molecular composition of the brain have been poorly understood. For this reason, there needs to be new innovations in brain mapping pursuits, and expansion microscopy (ExM) is proposed throughout this thesis as a potential candidate for most effectively meeting the needs of the efforts. First introduced in 2015, ExM allows for nanometer scale resolution to be achieved on a conventional microscope. By constructing an ​ expanding polymer network inside the biological specimen, conjugating the biomolecules of interest to the matrix, and letting it expand after getting rid of everything else we are not interested in imaging, the physical distance between the biomolecules anchored to the polymer matrix increases, effectively overcoming the diffraction-limit of the conventional confocal microscope and thereby increasing the effective resolution of the microscope down to nanometer scale. Over the course of my graduate studies, I worked on three improvements to this modality: (1) applying ExM iteratively to the specimen and increase the effective resolution exponentially, (2) developing iii intercalating lipid probes for visualizing lipid membranes in the context of ExM, and (3) devising a ExM-compatible approach to visualize extracellular space of a whole larval zebrafish. In addition to these, in an effort to understand what type of infrastructural help is needed to map the brain within our foreseeable future, I summarized an overview of current practices pursued by governments, industry, and academia to achieve scientific discoveries towards the end of this thesis. iv Table of Contents Introduction ------------------------------------------------------------------------------------------------------------------------- 1 Expansion Microscopy ----------------------------------------------------------------------------------------------------------- 9 ​ ​ Cookbook Style Expansion Microscopy Protocol ------------------------------------------------------------- 11 ​ ​ Materials List -------------------------------------------------------------------------------------------------------- 13 ​ ​ Protocol Steps ------------------------------------------------------------------------------------------------------ 17 ​ ​ Reagents and Solutions ----------------------------------------------------------------------------------------- 25 ​ ​ Iterative Expansion Microscopy ---------------------------------------------------------------------------------------------- 30 ​ ​ Resolution Validation for Expansion Microscopy ---------------------------------------------------------- 34 ​ ​ Membrane Expansion Microscopy (MxM) --------------------------------------------------------------------------------- 40 ​ ​ Intercalating Lipid Staining Probes: palm-GeLy ------------------------------------------------------------ 41 ​ ​ Immunohistochemistry-compatible MxM --------------------------------------------------------------------- 49 ​ ​ Directly Anchoring Lipids to ExM Gel ------------------------------------------------------------------------- 53 ​ ​ Iterative MxM (iMxM) ---------------------------------------------------------------------------------------------- 54 ​ ​ iMxM with Immunohistochemistry ------------------------------------------------------------------------------ 56 ​ ​ MxM with Fluorescent in-situ Hybridization for RNA imaging ------------------------------------------- 58 ​ ​ Methods -------------------------------------------------------------------------------------------------------------- 61 ​ ​ Extracellular Space Labeling of the Zebrafish using Expansion Microscopy ------------------------------------ 68 ​ ​ Whole Larval Zebrafish Expansion Microscopy ------------------------------------------------------------ 69 ​ ​ Extracellular Space Labeling with Whole Larval Zebrafish Expansion ------------------------------- 71 ​ ​ Bridging the Gap between Research and Impact: Past and Future ------------------------------------------------ 76 ​ ​ Government Agencies ------------------------------------------------------------------------------------------- 80 ​ ​ Accelerator Programs (Private Sector) ----------------------------------------------------------------------- 84 ​ ​ Academic Institutions --------------------------------------------------------------------------------------------- 86 ​ ​ Conclusion ----------------------------------------------------------------------------------------------------------------------- 100 ​ ​ Bibliography --------------------------------------------------------------------------------------------------------------------- 102 ​ ​ v Acknowledgement Without Professor Ed Boyden, I wouldn’t be writing this thesis right now. When I first emailed Ed for a meeting back in 2015, I was on the verge of quitting my PhD for various personal reasons. During our short meeting (I was 25 minutes late to our 30 minute meeting because I am bad with directions and MIT buildings are, still, so confusing to me), he not only inspired me to continue my PhD, but also allowed me to join his neurobiology lab even though I had no prior experience in this field. He then went through all the trouble, so that I could work in his MIT lab as a Harvard student. Really, I owe Ed billion thanks. Professor Jennifer Lewis, my Harvard academic advisor, has also been extremely generous to me. She had no practical reason to be my academic advisor, but she volunteered and saved my academic career. Professor Federico Capasso and John Girash were also there for me when I had to figure out the complications involved in working at MIT as a Harvard student. More recently, Professor George Church and Professor Samir Mitragotri so willingly agreed to be my committee members, and I couldn’t thank them more. These are the people that made me rethink about altruism, and I am sincerely grateful for their support. And there are Manos Karagiannis and Jae-Byum Chang. Postdocs in our group who so patiently walked me through the world of bioengineering research on a daily basis. They taught me the practical know-hows and theoretical insights involved in every step of our projects. In addition to Manos and Jae-Byum, many members of our lab have been amazingly helpful on a personal and professional level: Anu, Nick, Monique, Jay, Fei, vi Cristina, Daniel, Alexi, Tay, Dan, Oz, Adam, to name a few. We spent many sleepless nights and fun-filled days together, and I am very lucky to be able to call them both my colleagues and friends. Outside the lab, I want to extend my gratitude to my supportive partner Rachel. Finally, I would like to thank Samsung Scholarship for supporting my graduate studies and my parents Shichul Kang and Hyewon Oh, as well as my brother Mok Kang and our beloved canine sister Poolibi, for all their infinite support. As for my next career, I will be working with Joichi Ito and Jessica Traynor on a new initiative, and I also owe them billion thanks for making my transition and my new journey possible. Hope I can make everyone who believes in me proud. I will try my best! vii Expansion Microscopy for Brain Mapping Chapter 1 Introduction More than one billion people in the world suffer from brain disorders, inflicting 1 in 6 of the world’s populations. More than one trillion US dollars are spent on addressing these, but the results have been discouraging. There were 267 programs in the portfolios of large pharmaceutical companies (i.e., Sanofi/Genzyme, Novartis, Johnson & Johnson, etc) in 2010, but five years after, that number has decreased to 129. It costs more than one billion dollars to develop the drug, but during the process of clinical trials mandated 1,2 by Food and Drug Administration, ~92% fail to receive clinical approval .​ For example, ​ just a few weeks ago, Biogen announced its ending of Alzheimer’s drug trials after years 3 of hard work, and as a result, its stock prices plunged by 26% .​ We see people suffering ​ from brain disorders within our
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