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Whole-Exm: Expansion Microscopy Imaging of All Anatomical Structures

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Whole-Exm: Expansion Microscopy Imaging of All Anatomical Structures bioRxiv preprint doi: https://doi.org/10.1101/2021.05.18.443629; this version posted May 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Whole-ExM: Expansion microscopy imaging of all 2 anatomical structures of whole larval zebrafish 3 4 Jueun Sim1,18, Chan E Park1,18, In Cho1,18, Kyeongbae Min2, Jeong-Soo Lee3,4,5, Yosep Chong6, Jeewon 5 Kim7, Jeong Seuk Kang8, Kiryl D. Piatkevich9,10,11, Erica E. Jung12, Seok-Kyu Kwon13,14, Young-Gyu Yoon7, 6 Edward S. Boyden15,16,17, and Jae-Byum Chang1* 7 8 1Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, 9 Daejeon, Republic of Korea 10 2Department of Biomedical Engineering, Sungkyunkwan University, Suwon, Republic of Korea 11 3Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, 12 Daejeon, Republic of Korea 13 4KRIBB School, University of Science and Technology, Daejeon, Republic of Korea 14 5Convergence Research Center for Dementia, Korea Institute of Science and Technology, Seoul, Republic 15 of Korea 16 6Department of Hospital Pathology, Uijeongbu St. Mary's Hospital, College of Medicine, The Catholic 17 University of Korea, Uijeongbu, Republic of Korea 18 7School of Electrical Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic 19 of Korea 20 8John A. Paulson School of Engineering and Applied Sciences Harvard University, Cambridge, MA, USA 21 9School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China 22 10Westlake Laboratory of Life Sciences and Biomedicine, Westlake University, Hangzhou, Zhejiang, China 23 11Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China 24 12Department of Mechanical and Industrial Engineering, The University of Illinois at Chicago, Chicago, IL, 25 USA 26 13Center for Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology, 27 Seoul, Republic of Korea 28 14Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and 29 Technology, Seoul, Republic of Korea 30 15Howard Hughes Medical Institute, Cambridge, MA, USA. 31 16McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA. 32 17Departments of Brain and Cognitive Sciences, Media Arts and Sciences, and Biological Engineering, bioRxiv preprint doi: https://doi.org/10.1101/2021.05.18.443629; this version posted May 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 33 Massachusetts Institute of Technology, Cambridge, MA, USA. 34 18These authors contributed equally: Jueun Sim, Chan E Park, and In Cho 35 *Corresponding Author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.05.18.443629; this version posted May 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 ABSTRACT 37 Nanoscale imaging of all anatomical structures over whole vertebrates is needed for a systematic 38 understanding of human diseases, but this has not yet been achieved. Here, we demonstrate whole-ExM, 39 which enables nanoscale imaging of all anatomical structures of whole zebrafish larvae by labeling the 40 proteins of the larvae with fluorophores and expanding them four-fold. We first optimize the fluorophore 41 selection and labeling procedure to visualize a broader range of anatomical structures. We then develop an 42 expansion protocol for zebrafish larvae having calcified body parts. Through this process, we visualize the 43 nanoscale details of diverse larvae organs, which have corresponding organ counterparts in humans, over 44 the intact larvae. We show that whole-ExM retains the fluorescence signals of fluorescent proteins, and its 45 resolution is high enough to visualize various structures that can be imaged only with electron microscopy. 46 Whole-ExM would enable the nanoscale study of the molecular mechanisms of human diseases. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.18.443629; this version posted May 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 INTRODUCTION 48 A systematic understanding of biological systems requires an unbiased investigation of the whole organism 49 at sub-cellular resolutions1. In developmental biology, for instance, a high spatial resolution is essential for 50 discerning differences in subcellular structures or morphological details during different developmental 51 stages, whereas full spatial coverage is needed for the quantitative analysis of organ morphogenesis from a 52 systematic persepective2. Various imaging techniques have been developed to visualize whole organism, 53 including tissue clearing3–5, computed tomography6, and electron microscopy7. Computed tomography and 54 tissue clearing do not rely on physical sectioning and this allow for non-invasive, three-dimensional 55 histological analysis6,8. Nonetheless, these approaches do not provide sub-cellular resolution beyond 56 millimeter-scales with a sufficiently high contrast to distinguish sub-cellular structures6. Electron 57 microscopy has been combined with automated sectioning to facilitate volumetric tissue imaging at a 58 nanoscale resolution9,10; however, sample preparation and image reconstruction require specialized 59 instruments and skilled personnel for the imaging of a whole organism11. Simple and straightforward 60 approaches for imaging intact organisms at a subcellular resolution remain an unmet need in biology. 61 Expansion microscopy (ExM) combined with the labeling of functional groups of specimens with 62 fluorophores would be an attractive candidate for satisfying such a need12–16. ExM can image proteins or 63 mRNA in diverse specimens with a lateral resolution of 60-nm by physically expanding the specimens17–23. 64 Recently developed ExM techniques utilizing fluorophore labeling have enabled the unbiased visualization 65 of nanoscale details of subcellular structures and organelles by labeling various types of molecules, such as 66 proteins12,14,24, nucleic acids12,16, or lipids15,16, without antibodies12–14,16. However, the application of ExM 67 to whole organisms has been limited to relatively small and simple organisms, such as bacteria25–27, fungi28, 68 and parasitic micro-organisms29. Recently, whole-organism ExM imaging of C. elegans has been 69 demonstrated14,30. However, ExM imaging of whole vertebrates, which share a high degree of genetic and 70 anatomical similarities with humans, has not yet been demonstrated because of the difficulty of expanding 71 hard body parts, such as bones. 72 In this paper, we demonstrate a new ExM technique, whole-ExM, which can visualize all anatomically 73 relevant structures of whole larval zebrafish. We first show that the range of structures visualized by 74 fluorophore N-Hydroxysuccinimide (NHS)-ester staining depends on the hydrophobicity of the 75 fluorophores used. Thus, we use multiple fluorophore NHS esters with different hydrophobicities to reveal 76 all major anatomically relevant cellular structures and cellular organelles that could not be observed with 77 only one fluorophore NHS ester. Then, we show that even more diverse structures can be visualized when 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.18.443629; this version posted May 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 78 fluorophore NHS esters are applied in the middle of the digestion process compared with when they are 79 applied before or after digestion. Finally, we optimize the ExM protocol for vertebrates in which bones and 80 soft tissues are entangled. By combining these three approaches, we visualize all anatomically relevant 81 structures of whole larval zebrafish with a lateral resolution of 60-nm without the need for any antibodies 82 or a thin-slicing process (see Supplementary Video 1 for the whole-ExM imaging of a zebrafish larva). 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.18.443629; this version posted May 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 83 RESULTS 84 Effect of the physicochemical properties of fluorophores on the range of labeled structures 85 We first investigated whether all anatomically relevant structures could be visualized using fluorophore 86 NHS-ester staining. Recently developed expansion microscopy techniques visualize diverse cellular 87 structures and cellular organelles by conjugating fluorophores to functional groups of specific molecular 88 types12–14,16, such as the conjugation of fluorophore NHS esters to the primary amines of proteins12–14. 89 However, the effect of fluorophores on the range of structures that can be visualized has not been studied. 90 We hypothesized that the protein structures labeled with fluorophore NHS esters would depend on the 91 physicochemical properties of the fluorophores, especially their hydrophobicity. To verify this hypothesis, 92 mouse brain slices were
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