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Expansion microscopy at one nanometer resolution

Ali H. Shaib, Abed Alrahman Chouaib, Vanessa Imani, Rajdeep Chowdhury, Svilen Veselinov Georgiev, Nikolaos Mougios, Mehar Monga, Sofiia Reshetniak, Daniel Mihaylov, Han Chen, Parisa Fatehbasharzad, Dagmar Crzan, Kim-Ann Saal, Claudia Trenkwalder, Brit Mollenhauer, Tiago F. Outeiro, Julia Preobraschenski, View ORCID ProfileUte Becherer, View ORCID ProfileTobias Moser, Edward S. Boyden, A Radu Aricescu, Markus Sauer, View ORCID ProfileFelipe Opazo, Silvio O. Rizzoli
doi: https://doi.org/10.1101/2022.08.03.502284
Ali H. Shaib
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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  • For correspondence: ali.shaib@med.uni-goettingen.de srizzol@gwdg.de
Abed Alrahman Chouaib
2Department of Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg, Germany
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Vanessa Imani
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Rajdeep Chowdhury
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Svilen Veselinov Georgiev
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Nikolaos Mougios
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
3Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany
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Mehar Monga
4Biochemistry of Membrane Dynamics Group, Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany
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Sofiia Reshetniak
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Daniel Mihaylov
5MRC Laboratory of Molecular Biology, Cambridge, UK
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Han Chen
6Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany
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Parisa Fatehbasharzad
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Dagmar Crzan
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Kim-Ann Saal
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
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Claudia Trenkwalder
7Department of Neurosurgery, University Medical Center, Göttingen, Germany
8Paracelsus-Elena-Klinik, Kassel, Germany
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Brit Mollenhauer
8Paracelsus-Elena-Klinik, Kassel, Germany
9Department of Neurology, University Medical Center, Göttingen, Germany
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Tiago F. Outeiro
10Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, 37073 Göttingen, Germany; Max Planck Institute for Multidisciplinary Sciences, 37075 Göttingen, Germany; Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, NE2 4HH, United Kingdom; Scientific Employee with an Honorary Contract at German Center for Neurodegenerative Diseases (DZNE), 37075 Göttingen, Germany
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Julia Preobraschenski
4Biochemistry of Membrane Dynamics Group, Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany
11Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Goettingen, Göttingen, Germany
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Ute Becherer
2Department of Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg, Germany
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  • ORCID record for Ute Becherer
Tobias Moser
6Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany
11Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Goettingen, Göttingen, Germany
12Auditory Neuroscience and Synaptic Nanophysiology Group, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
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Edward S. Boyden
13Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA; McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA; Koch Institute, Massachusetts Institute of Technology, Cambridge, MA, USA; Center for Neurobiological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
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A Radu Aricescu
5MRC Laboratory of Molecular Biology, Cambridge, UK
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Markus Sauer
14Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
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Felipe Opazo
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
3Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany
15NanoTag Biotechnologies GmbH, Rudolf-Wissell-Straße 28a, 37079 Göttingen, Germany
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Silvio O. Rizzoli
1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany
3Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany
11Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Goettingen, Göttingen, Germany
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  • For correspondence: ali.shaib@med.uni-goettingen.de srizzol@gwdg.de
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Abstract

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm2,3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4,5. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6,7 to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

Competing Interest Statement

S.O.R. and F.O. are shareholders of NanoTag Biotechnologies GmbH. The remaining authors declare no competing interests. E.S.B. is an inventor on multiple patents related to expansion microscopy, and co-founder of a company working on commercial applications of expansion microscopy.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Expansion microscopy at one nanometer resolution
Ali H. Shaib, Abed Alrahman Chouaib, Vanessa Imani, Rajdeep Chowdhury, Svilen Veselinov Georgiev, Nikolaos Mougios, Mehar Monga, Sofiia Reshetniak, Daniel Mihaylov, Han Chen, Parisa Fatehbasharzad, Dagmar Crzan, Kim-Ann Saal, Claudia Trenkwalder, Brit Mollenhauer, Tiago F. Outeiro, Julia Preobraschenski, Ute Becherer, Tobias Moser, Edward S. Boyden, A Radu Aricescu, Markus Sauer, Felipe Opazo, Silvio O. Rizzoli
bioRxiv 2022.08.03.502284; doi: https://doi.org/10.1101/2022.08.03.502284
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Expansion microscopy at one nanometer resolution
Ali H. Shaib, Abed Alrahman Chouaib, Vanessa Imani, Rajdeep Chowdhury, Svilen Veselinov Georgiev, Nikolaos Mougios, Mehar Monga, Sofiia Reshetniak, Daniel Mihaylov, Han Chen, Parisa Fatehbasharzad, Dagmar Crzan, Kim-Ann Saal, Claudia Trenkwalder, Brit Mollenhauer, Tiago F. Outeiro, Julia Preobraschenski, Ute Becherer, Tobias Moser, Edward S. Boyden, A Radu Aricescu, Markus Sauer, Felipe Opazo, Silvio O. Rizzoli
bioRxiv 2022.08.03.502284; doi: https://doi.org/10.1101/2022.08.03.502284

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