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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

Universal Molecular Retention with 11-Fold Expansion Microscopy

Published: October 6th, 2023

DOI:

10.3791/65338

1Department of Biological Sciences, Carnegie Mellon University

Presented here is a new version of expansion microscopy (ExM), Magnify, that is modified for up to 11-fold expansion, conserving a comprehensive array of biomolecule classes, and is compatible with a broad range of tissue types. It enables the interrogation of the nanoscale configuration of biomolecules using conventional diffraction-limited microscopes.

The nanoscale imaging of biological specimens can improve the understanding of disease pathogenesis. In recent years, expansion microscopy (ExM) has been demonstrated to be an effective and low-cost alternative to optical super-resolution microscopy. However, it has been limited by the need for specific and often custom anchoring agents to retain different biomolecule classes within the gel and by difficulties with expanding standard clinical sample formats, such as formalin-fixed paraffin-embedded tissue, especially if larger expansion factors or preserved protein epitopes are desired. Here, we describe Magnify, a new ExM method for robust expansion up to 11-fold in a wide array of tissue types. By using methacrolein as the chemical anchor between the tissue and gel, Magnify retains multiple biomolecules, such as proteins, lipids, and nucleic acids, within the gel, thus allowing the broad nanoscale imaging of tissues on conventional optical microscopes. This protocol describes best practices to ensure robust and crack-free tissue expansion, as well as tips for handling and imaging highly expanded gels.

Biological systems exhibit structural heterogeneity, from the limbs and the organs down to the levels of proteins at the nanoscale. Therefore, a complete understanding of the operation of these systems requires visual examination across these size scales. However, the diffraction limit of light causes challenges in visualizing structures smaller than ~200-300 nm on a conventional fluorescence microscope. In addition, optical super-resolution methods1,2,3, such as stimulated emission depletion (STED), photo-activated localization microscopy (PALM), stochastic optical reco....

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All the experimental procedures involving animals were conducted in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Institutional Animal Care and Use Committee at Carnegie Mellon University. Human tissue samples were commercially obtained.

1. Preparation of the stock reagents and solutions

NOTE: Refer to the Table of Materials for a list of the reagents used.

  1. Prepare the gellin.......

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If the protocol has been successfully completed (Figure 1), the sample will appear clear and flat after heat denaturation; any folding or wrinkling indicates incomplete homogenization. A successfully expanded sample will be 3-4.5-fold larger than before expansion in 1x PBS and 8-11-fold larger when fully expanded in ddH2O. Figure 3 shows example pre- and post-expansion images of 5 µm thick FFPE human kidney sample processed using this protocol an.......

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Here, we present the Magnify protocol17, an ExM variant that can retain multiple biomolecules with a single chemical anchor and expand challenging FFPE clinical specimens up to 11-fold with heat denaturation. The key changes in this protocol that distinguish it from other ExM protocols include the use of a reformulated gel that remains mechanically robust even when fully expanded, as well as the use of methacrolein as the biomolecule anchor. The most critical steps in this protocol are as follows:.......

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This work was supported by Carnegie Mellon University and the D.S.F. Charitable Foundation (Y.Z. and X.R.), the National Institutes of Health (N.I.H.) Director's New Innovator Award DP2 OD025926-01, and the Kauffman Foundation.

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Name Company Catalog Number Comments
4-hydroxy-TEMPO (4HT) Sigma Aldrich 176141 Inhibitor
6-well glass-bottom plate (#1.5 coverglass) Cellvis P06-1.5H-N
Acrylamide Sigma Aldrich A8887 Gel Monomer component
Ammonium persulfate (APS)  Sigma Aldrich A3678 Initiatior
DAPI (1 mg/mL) Thermo Scientific 62248
Decaethylene glycol mono dodecyl ether (C12E10) Sigma Aldrich P9769 Non-ionic surfactant
Diamond knife No. 88 CM General Tools 31116
Ethanol Pharmco 111000200
Ethanol Pharmco 111000200
Ethylenediaminetetraacetic
acid (EDTA) 0.5 M
VWR BDH7830-1 Homogenization Buffer Component
Forceps
Glycine Sigma Aldrich G8898 Homogenization Buffer Component
Heparin Sigma Aldrich H3393
Methacrolein Sigma Aldrich 133035 Anchoring Agent
Micro cover Glass #1 (24x60mm) VWR 48393 106
Micro cover Glass #1.5 (24x60mm) VWR 48393 251
N,N,N′,N′-
Tetramethylethylenediamine (TEMED)
Sigma Aldrich T9281 Accelerator
N,N′-Methylenebisacrylamide (Bis) Sigma Aldrich M7279 Gel Monomer component
N,N-dimethylacrylamide (DMAA) Sigma Aldrich 274135 Gel Monomer component
Nunclon 4-Well x 5 mL MultiDish Cell Culture Dish Thermo Fisher 167063
Nunclon 6-Well Cell Culture Dish Thermo Fisher 140675
Nunc™ 15mL Conical Thermo Fisher 339651
Nunc™ 50mL Conical Thermo Fisher 339653
Orbital Shaker
Paint brush
pH Meter
Phosphate Buffered Saline (PBS), 10x Solution Fischer Scientific BP399-1
Polyethylene glycol  200 Sigma Aldrich P-3015
Proteinase K (Molecular Biology Grade) Thermo Scientific EO0491
Razor blade Fischer Scientifc 12640
Safelock Microcentrifuge Tubes 1.5 mL Thermo Fisher 3457
Safelock Microcentrifuge Tubes 2.0 mL Thermo Fisher 3459
Sodium acrylate (SA) AK Scientific R624 Gel Monomer component
Sodium azide Sigma Aldrich S2002
Sodium chloride Sigma Aldrich S6191
Sodium citrate tribasic dihydrate Sigma Aldrich C8532-1KG
Sodium dodecyl sulfate (SDS) Sigma Aldrich L3771 Homogenization Buffer Component
Tris Base Fischer Scientific BP152-1 Homogenization Buffer Component
Triton X-100 Sigma Aldrich T8787
Urea Sigma Aldrich U5378 Homogenization Buffer Component
Xylenes Sigma Aldrich 214736
20x SSC Thermo Scientific AM9763
Tween20 Sigma Aldrich P1379
poly-L-lysine  Sigma Aldrich P8920

  1. Hell, S. W. Far-field optical nanoscopy. Science. 316 (5828), 1153-1158 (2007).
  2. Combs, C. A., Shroff, H. Fluorescence microscopy: A concise guide to current imaging methods. Current Protocols in Neuroscience. 79, 1-25 (2017).
  3. Schermelleh, L., Heintzmann, R., Leonhardt, H. A guide to super-resolution fluorescence microscopy. Journal of Cell Biology. 190 (2), 165-175 (2010).
  4. Chen, F., Tillberg, P. W., Boyden, E. S. Expansion microscopy. Science. 347 (6221), 543-548 (2015).
  5. Tillberg, P. W., et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nature Biotechnology. 34 (9), 987-992 (2016).
  6. Chozinski, T. J., et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nature Methods. 13 (6), 485-488 (2016).
  7. Ku, T., et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nature Biotechnology. 34 (9), 973-981 (2016).
  8. Chen, F., et al. Nanoscale imaging of R.N.A. with expansion microscopy. Nature Methods. 13 (8), 679-684 (2016).
  9. Tsanov, N., et al. SmiFISH and FISH-quant - A flexible single R.N.A. detection approach with super-resolution capability. Nucleic Acids Research. 44 (22), 165 (2016).
  10. Wang, G., Moffitt, J. R., Zhuang, X. Multiplexed imaging of high-density libraries of R.N.A.s with MERFISH and expansion microscopy. Scientific Reports. 8 (1), 4847 (2018).
  11. Wen, G., et al. Evaluation of direct grafting strategies via trivalent anchoring for enabling lipid membrane and cytoskeleton staining in expansion microscopy. ACS Nano. 14 (7), 7860-7867 (2020).
  12. Sun, D., et al. Click-ExM enables expansion microscopy for all biomolecules. Nature Methods. 18, 107-113 (2021).
  13. White, B. M., Kumar, P., Conwell, A. N., Wu, K., Baskin, J. M. Lipid expansion microscopy. Journal of the American Chemical Society. 144 (40), 18212-18217 (2022).
  14. Truckenbrodt, S., et al. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Reports. 19 (9), e45836 (2018).
  15. Chang, J. -. B., et al. Iterative expansion microscopy. Nature Methods. 14 (6), 593-599 (2017).
  16. Park, J., et al. Epitope-preserving magnified analysis of proteome (eMAP). Science Advances. 7 (46), (2021).
  17. Klimas, A., et al. Magnify is a universal molecular anchoring strategy for expansion microscopy. Nature Biotechnology. , (2023).
  18. Gao, M., et al. Expansion stimulated emission depletion microscopy (ExSTED). ACS Nano. 12 (5), 4178-4185 (2018).
  19. Zwettler, F. U., et al. Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Nature Communications. 11, 3388 (2020).

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