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&#39;s New Innovator Award DP2 OD025926-01, and the Kauffman Foundation.

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.
<ol>
	<li>Prepare the gellin...

Expansion Microscopy for Robust 11-Fold Expansion

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

The authors declare the following competing financial interest(s): Y.Z., A.K., Z.C., and B.R.G. invented several inventions related to Magnify and ExM.

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:...

Biological systems&#160;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 reconstruction microscopy (STORM), and structured illumination microscopy (SIM), though powerful, present their own challenges, as they require expensive hardware and reagents and often have slow acquisition times and a poor ability to image large volumes in 3D.
Expansion microscopy4 (ExM) provides an alternative means of circumventing the diffraction limit of light by covalently anchoring biomolecules into a water-swellable polymer gel and physically pulling them apart, thus rendering them resolvable on conventional optical microscopes. A multitude of ExM protocol variants have been developed since the original publication of&#160;ExM less than a decade ago, and these protocols allow the direct incorporation of proteins5,6,7, RNA8,9,10, or lipids11,12,13 into the gel network by altering the chemical anchor or expanding the sample further (thus improving the effective resolution) either in a single step14 or multiple iterative steps15,16. Until recently, no single ExM protocol could retain these three biomolecule classes with a single commercially available chemical anchor while providing a mechanically sturdy gel that could expand ~10-fold in a single expansion round.
Here, we present Magnify17, a recent addition to the ExM arsenal that uses methacrolein as the biomolecule anchor. Methacrolein forms covalent bonds with tissue like that of paraformaldehyde, ensuring that multiple classes of biomolecules can be retained within the gel network without requiring various specific or custom anchoring agents. Additionally, this technique can expand a broad spectrum of tissues up to 11-fold, including notoriously challenging samples such as formalin-fixed paraffin-embedded (FFPE) clinical samples. Previous methods for expanding such mechanically rigid samples required harsh protease digestion, rendering the antibody labeling of proteins of interest impossible after the sample had been expanded. In contrast, this technique achieves the expansion of FFPE clinical samples using a hot denaturing solution, thus preserving whole protein epitopes within the gel, which can be targeted for post-expansion imaging (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-hydroxy-TEMPO (4HT)</td>
			<td>Sigma Aldrich</td>
			<td>176141</td>
			<td>Inhibitor</td>
		</tr>
		<tr>
			<td>6-well glass-bottom plate (#1.5 coverglass)</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma Aldrich</td>
			<td>A8887</td>
			<td>Gel Monomer component</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A3678</td>
			<td>Initiatior</td>
		</tr>
		<tr>
			<td>DAPI (1 mg/mL)</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Decaethylene glycol mono dodecyl ether (C12E10)</td>
			<td>Sigma Aldrich</td>
			<td>P9769</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Diamond knife No. 88 CM</td>
			<td>General Tools</td>
			<td>31116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic 
			acid (EDTA) 0.5 M</td>
			<td>VWR</td>
			<td>BDH7830-1</td>
			<td>Homogenization Buffer Component</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>G8898</td>
			<td>Homogenization Buffer Component</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrolein</td>
			<td>Sigma Aldrich</td>
			<td>133035</td>
			<td>Anchoring Agent</td>
		</tr>
		<tr>
			<td>Micro cover Glass #1 (24x60mm)</td>
			<td>VWR</td>
			<td>48393 106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover Glass #1.5 (24x60mm)</td>
			<td>VWR</td>
			<td>48393 251</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;- 
			Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma Aldrich</td>
			<td>T9281</td>
			<td>Accelerator</td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebisacrylamide (Bis)</td>
			<td>Sigma Aldrich</td>
			<td>M7279</td>
			<td>Gel Monomer component</td>
		</tr>
		<tr>
			<td>N,N-dimethylacrylamide (DMAA)</td>
			<td>Sigma Aldrich</td>
			<td>274135</td>
			<td>Gel Monomer component</td>
		</tr>
		<tr>
			<td>Nunclon 4-Well x 5 mL MultiDish Cell Culture Dish</td>
			<td>Thermo Fisher</td>
			<td>167063</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon 6-Well Cell Culture Dish</td>
			<td>Thermo Fisher</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc&#8482; 15mL&#160;Conical</td>
			<td>Thermo Fisher</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc&#8482; 50mL&#160;Conical</td>
			<td>Thermo Fisher</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), 10x Solution</td>
			<td>Fischer Scientific</td>
			<td>BP399-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol&#160; 200</td>
			<td>Sigma Aldrich</td>
			<td>P-3015</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K (Molecular Biology Grade)</td>
			<td>Thermo Scientific</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fischer Scientifc</td>
			<td>12640</td>
			<td></td>
		</tr>
		<tr>
			<td>Safelock Microcentrifuge Tubes 1.5 mL</td>
			<td>Thermo Fisher</td>
			<td>3457</td>
			<td></td>
		</tr>
		<tr>
			<td>Safelock Microcentrifuge Tubes 2.0 mL</td>
			<td>Thermo Fisher</td>
			<td>3459</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acrylate (SA)</td>
			<td>AK Scientific</td>
			<td>R624</td>
			<td>Gel Monomer component</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8532-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>L3771</td>
			<td>Homogenization Buffer Component</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fischer Scientific</td>
			<td>BP152-1</td>
			<td>Homogenization Buffer Component</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>U5378</td>
			<td>Homogenization Buffer Component</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Thermo Scientific</td>
			<td>AM9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
	</tbody>
</table>

universal molecular retention with 11-fold expansion microscopy

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.

Author Spotlight: Universal Molecular Retention with 11-Fold Expansion Microscopy  

Universal Molecular Retention with 11-Fold Expansion Microscopy

We're trying to find easy and low cost ways for researchers to image their biological specimens at resolutions below the diffraction limit, particularly in the areas of neuroscience and clinical pathology. Many recent expansion microscopy developments involve modifying gel chemistry to expand samples further, changing the anchoring molecule to visualize different targets and utilizing heating alteration to preserve protein epitopes. With innovated magnify, a robust expansion microscopy method that allows nanoscale imaging of various biological or clinical specimens.It simplifies super resolution microscopy by eliminating specialized equipment needs while preserving intricate biological features with up to 15 nanometers resolution, hence increasing accessibility and utility in labs worldwide. Previously users had to experiment with different expansion microscopy techniques, depending on the tissue and biomolecules they wanted to image. Magnify enables high resolution imaging across various biomolecules and tissue types without requiring specific anchoring steps or specialized equipment, thus bridging a significant gap in nanoscale imaging.Magnify expands tougher clinical samples over fourfold while preserving protein epitopes enabling more accurate investigation of queuing disease. Its chemical anchor also preserves a wide range of biomolecules, making it useful for basic research in animal models Magnify simplifies nanoscale imaging of biomolecules and intact specimens without complex optics or specialized equipment. It's adaptable across various imaging modalities and expansion microscopy strategies, allowing accessible cost-effective nanoscale imaging that may accelerate drug treatments and disease studies while potentially transforming tissue atlas generation.Magnify offers endless possibilities for research. Current focus areas include expanding tissue site, investigating pathological human samples at the nanoscale and studying the smallest scale changes on brain during learning and disease.

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 &#181;m thick FFPE human kidney sample processed using this protocol an...

Watch this Scientific Journal Video about Universal Molecular Retention with 11-Fold Expansion Microscopy at JoVE.com

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 ...

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2.2K Views.  Carnegie Mellon University. Stiamo cercando di trovare modi semplici e a basso costo per i ricercatori di visualizzare i loro campioni biologici a risoluzioni inferiori al limite di diffrazione, in particolare nelle aree delle neuroscienze e della patologia clinica. Molti recenti sviluppi della microscopia ad espansione comportano la modifica della chimica del gel per espandere ulteriormente i campioni, cambiando la molecola di ancoraggio per visualizzare diversi bersagli e utilizzando l'alterazione del riscaldamento pe...