Expansion Microscopy: Scalable and Multiplexed Nanoscale Imaging by Fei Chen B.S. Electrical Engineering California Institute of Technology, 2011 Submitted to the Department of Biological Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering at the Massachusetts Institute of Technology February 2017 2017 Massachusetts Institute of Technology. All rights reserved. Signature of author Signature redacted Department of Biological Engineering 2017 Certified bj Sr rd February, 22, Signature redacted_____ - Edward S. Boyden Professor of Biological Engineering, Brain and Cognitive Sciences, Media Arts and Sciences Thesis Supervisor AcceptedbySig ature redacted Mark Bathe MASSACHUSETTS INSTITUTE Professor of Biological Engineering OF TECHNOLOGY (/ Chair, Graduate Program Committee LU MAY 0 9 2017 LIBRARIES Thesis Committee Members Peter T. C. So, Ph.D. Professor of Biological Engineering and Mechanical Engineering Thesis Committee Chair Scott R. Manalis, Ph.D. Professor of Biological Engineering and Mechanical Engineering Thesis Committee Member Edward S. Boyden, Ph.D. Professor of Biological Engineering, Brain and Cognitive Sciences, Media Arts and Sciences Thesis Supervisor Table of Contents A B S T R A C T ................................................................................ ------ .... ......----------. .. .. - I ACKNOWLEDGEMENTS..................................................................................................... 2 CHAPTER ONE: INTRODUCTION.......................................... 4 SUPER-RESOLUTION M ICROSCOPY............................................................ ....- --- ---........................................................... 4 EXPANSION M ICROSCOPY........................................................................---------............................................................5 CITATIONS TO PUBLISHED WORK AND ACKNOWLEDGEMENTS TO CO-A UTHORS ....................................................................... 8 CHAPTER TWO: EXPANSION MICROSCOPY, A SCALABLE SUPER-RESOLUTION MICROSCOPY PLATFORM ........................................................................................... 9 RESULTS.............. .............................................................................-- - -......------- DISCUSSION.................................................................................... ------------............................................................ 12 FIGURES............ ................................................................................ - -- - - - - - - ... 14 Figure 1-1: Expansion microscopy (ExM) concept. ......................................... 14 Figure 1-2: Expansion microscopy physically magnifies with nanoscale isotropy, enabling super-resolution imaging on diffraction-limited microscopes..............................................15 Figure 1-3: Super-resolution imaging of synapses and neurons in intact mammalian brain tissue using ExM. 17 Figure 1-4: Scalable 3D super-resolution microscopy of mouse brain tissue. ....................... 19 SUPPLEM ENTARY INFORM ATIO N ...................................................-- ........... .... -------------------------------------- 20 M ETHODS ............................................ .. .. .. ... ...- ..---- ----...- -------..--- --.-- ----............................................................28 Labels for ExM :......................................................................................................... ---. -....--. ------.................... 28 Cultured cell preparation and staining:.................................................-28 Brain tissue preparation and staining:.............................................. -- 29 In situ polym er synthesis: ..............................................................................---- ..... --.----.................... 29 D igestion and expansion:..................................................................................... ...... ----.-. ----.-.-.-.................. 30 Optical clearing m easurements: .........................................................................................---.-.. ...- ---.-.......... 31 CHAPTER THREE: PROTEIN RETENTION EXPANSION MICROSCOPY......................... 34 INTRODUCTION........................................................................ ....- - - -- - - -- - --............................................................34 RESULTS............................................................................................................................... 34 DISCUSSION ................................................................... .....--.. -.-.-.--.-------.-----........................................................... 38 FIGURES ................................................................................................................................. 40 Figure 3-1. Post-expansion antibody delivery, after epitope-preserving homogenization..... .......... 40 Figure 3-2. Retention offluorescent protein (FP) and antibody fluorescence signals in proExM and pro ExM of FP fusions. ............................................................................-- .......... ..---. -...............................42 Figure 3-3. Validation of proExM in different marnmalian tissue types...........................44 Figure 3-4. proExM of mammalian brain circuitry. ....................................... .46 Figure 3-5. Workflows for expansion microscopy with protein retention. ................................................ 48 SUPPLEMENTARY INFORMATION ..................................................... .... ........ ............................................................ 49 M ETHODS...........................................- ................ ..-.----...... .- ..- - --............................................................ 64 CHAPTER FOUR: NANOSCALE IMAGING OF RNA WITH EXPANSION MICROSCOPY ....................................................................................................... 71 INT RO D U CT IO N ............................................................................................................................................................ 7 1 R ESU LTS ..................................................................................................................................................................... 71 D ISC U SSIO N ................................................................................................................................................................ 75 F IG U RES ..................................................................................................................................................................... 77 Figure 4-1. Design and validation of ExFISH chemistry. ..................................................................................... 77 Figure 4-2. Serially hybridized and m ultiplexed ExFISH ...................................................................................... 78 Figure 4-3. Nanoscale imaging of RNA in mammalian brain. ............................................................................ 79 SUPPLEMENTARY INFORMATION ..................................................................................................................................... 80 M ET H O D S .................................................................................................................................................................. 9 2 R EFER EN C ES ............................................................................................................................. 99 Abstract Microscopy has facilitated the discovery of many biological insights by optically magnifying small structures in cells and tissues. However, the resolution of optical microscopy is limited by the diffraction of light to ~200-300 nm, comparable or larger to the size of many subcellular structures. In this thesis, we describe a suite of tools based on a novel super-resolution microscopy approach called Expansion microscopy. Expansion microscopy (ExM) physically expands tissues so that the resolution of ordinary microscopes is increased -5 times by leveraging the swelling properties of polyelectrolyte hydrogels. Ordinary microscopes used with ExM are more accessible and faster than the specialized optical systems designed to image beyond the diffraction limit (e.g., STORM/PALM, STED, SIM), while yielding similar performance. Expanded tissues are also optically clear, allowing for unprecedented super-resolution imaging in thick tissues and facile reagent diffusion into the sample. We have since developed a variant of ExM, called protein retention ExM, in which proteins are directly anchored to the swellable gel using a commercially available cross-linking molecule. This strategy enables ExM of genetically encoded fluorescent proteins and commercial fluorescently labeled secondary antibodies. With these advancements, ExM can be carried out with purely commercial reagents and represents a simple extension of standard histological methods used to prepare samples for imaging. Furthermore, we have developed a variant of the ExM technology that enables RNA molecules to be directly linked to the ExM gel network via a small molecule linker and isotropic expansion. This technology, termed ExFISH, enables visualization of RNAs with nanoscale precision and single molecule resolution. We have demonstrated