This work was supported by the U.S. National Institutes of Health (R00 GM126136 to X.S.), the U.S. National Science Foundation (DMS1763272 to S.P.) and the Simons Foundation (594598 to S.P.).

1. Cell culture
<ol>
	<li>Use U2OS cells cultured in McCoy&#39;s 5A medium supplemented with 10% FBS at 37 &#176;C in 5% CO2.</li>
	<li>Culture cells onto a 16 well removable chambered coverglass (culture area 0.4 cm2) for ease of handling.</li>
</ol>
2. Fixation and permeabilization
NOTE: Fixation and permeabilization conditions depend on the optimized immunostaining protocols. The following is a fixation and permeabilization protocol to co-immunostain microtubule and clathrin coated pits (CCPs).
<ol>
	<li>Once cell counts reach ~0.04 x 106, fix cells with 100 &#181;L of 3.2% paraformaldehyde (PFA) in PEM buffer (100 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.9) for 10 min at room temperature (Table 1). 
	NOTE: Fixation using PFA is performed in a certified safety hood.</li>
	<li>Wash cells three times for 5 min each with 200 &#181;L of phosphate-buffered saline (PBS).</li>
	<li>Permeabilize cells with the 100 &#181;L of permeabilization buffer at room temperature for 15 min (Table 1). 
	&#8203;NOTE: Proceed with labeling as soon as possible to avoid target protein, RNA or DNA degradation, and to obtain an optimal result. If it is needed, however, fixed samples can be stored for an elongated time (up to a month) in a PBS buffer at 4 &#176;C. Replace any evaporated PBS and protect the sample from photobleaching.</li>
</ol>
3. Endogenous streptavidin and biotin blocking
<ol>
	<li>Incubate cells with the streptavidin solution diluted in a cell blocking buffer (100 &#181;L) for 15 min at room temperature (Table 1).</li>
	<li>Rinse briefly with 200 &#181;L of cell blocking buffer.</li>
	<li>Incubate cells with 100 &#181;L of biotin solution diluted in a cell blocking buffer for 15 min at room temperature. 
	&#8203;NOTE: When using a self-labeling tagging approach, such as using SNAP- or CLIP- tags, skip step 4, and proceed to step 5.1: self labeling tagging approach.</li>
</ol>
4. Primary antibody staining
<ol>
	<li>Incubate cells with rat anti-&#945;-tubulin antibody (1:500 dilution) and rabbit anti-clathrin heavy-chain antibody (1:100 dilution) in 100 &#181;L of cell blocking buffer for 16 h at 4 &#176;C or 1 h at room temperature. Double the incubation time for the tissue samples.</li>
	<li>Wash samples four times with 200 &#181;L of PBS for 5 min each.</li>
</ol>
5. LR-ExM specific secondary antibody staining
<ol>
	<li>Conjugate IgG antibodies with the trifunctional linkers such as N-hydroxysuccinimide (NHS) - methacrylamide (MA) -Biotin or NHS-MA-Digoxigenin (Dig) (Figure 1B). Synthesis of trifunctional anchors and conjugation of the secondary antibodies are previously introduced in Shi et al.1. Self-labeling tagging approach: Conjugate IgG antibodies with the trifunctional linkers, such as MA- benzylguanine (BG)-Biotin or MA- benzylcytosine (BC)-Dig (Figure 1B). Synthesis of trifunctional anchors and a conjugation of the secondary antibodies are previously introduced in Shi et al.1.</li>
	<li>Incubate cells with the donkey anti-rabbit-Dig-MA (1:100 dilution) and donkey anti-rat-biotin-MA (1:100 dilution) secondary antibodies in 100 &#181;L of cell blocking buffer for 1 h at room temperature. Double this incubation time for the tissue samples.</li>
	<li>Wash samples four times in 200 &#181;L of PBS for 5 min each.</li>
</ol>
6. Additional anchoring
<ol>
	<li>Incubate cells with 0.25% glutaraldehyde (GA) in PBS (100 &#181;L) for 15 min at room temperature in order to anchor the proteins onto the hydrogel. Alternatively, use 25 mM methacrylic acid N-hydroxysuccinimide ester (MA-NHS) for anchoring. One hour incubation at room temperature is encouraged.</li>
	<li>Wash samples three times in PBS for 5 min each.</li>
</ol>
7. Gelation
NOTE: The expansion speed is determined by the diffusion time of salt and water out or into the gel; thus, casting thin gels speeds up expansion time.
<ol>
	<li>Remove the upper structure of the 16-well gel plate. Add 40 &#181;L of monomer solution to each well to condition cells on ice. Incubate on ice for 5 min.
	<ol>
		<li>To prepare a monomer solution, see Table 2.</li>
	</ol>
	</li>
	<li>Prepare the gelation solution on ice. Add double distilled water and 10% N,N,N&#8242;,N&#8242; Tetramethylethylenediamine (TEMED) accelerator to the monomer solution (10% TEMED: monomer solution = 1:47); do not add the initiator yet (Table 3).</li>
	<li>For a cell culture chamber with an area of 0.4 cm2, use a gelation solution volume of about 40 &#181;L. Prepare 45 &#181;L of gelation solution for each well.</li>
	<li>Add 10% ammonium persulfate (APS) to the gelation solution, incubate on ice, and immediately pipette 40 &#181;L of gelation solution into each silicone gasket well on ice. Incubate on ice for 3 min (10% APS:10% TEMED:monomer solution = 1:1:47).</li>
	<li>Protect the sample from light and move the coverglass in the 10 cm Petri dish to a 37 &#176;C incubator for 1.5 h for gelation. To keep the humidity of the gel during the 37 &#176;C incubation, cover the Petri dish with the lid, and put a few drops of water in the Petri dish.</li>
</ol>
8. Digestion
<ol>
	<li>After gelation, remove the glass to separate the gel and transfer the gel to 6 well plate. Digest embedded cells with 2 mL of digestion buffer (Table 1) overnight at room temperature or 4 h at 37 &#176;C. Use at least 10-fold excess volume of digestion buffer.</li>
	<li>Wash gels with at least a 10-fold excess volume of water than the final gel volume. Repeat the washing step four times for 20 to 30 min each time. The gel expands by about two-fold in each dimension. 
	&#8203;NOTE: Gel samples can be stored for up to 1 month in a PBS buffer at 4 &#176;C. Replace any evaporated water to avoid drying, and protect the sample from light during storage. To avoid degradation of trifunctional anchors, samples should be stained as soon as possible after digestion and wash.</li>
</ol>
9. Post-digestion fluorescence staining
<ol>
	<li>Incubate gels in 2 mL volume of streptavidin (STV)/digoxigenin (DIG) staining buffer with 2 to 5 &#181;M STV-dye and/or anti-DIG-dye for 24 h at room temperature. Keep the samples in the dark.</li>
</ol>
10. Expansion
<ol>
	<li>Wash and expand the gel four times with excessive water (2-3 mL) for 30 min to 1 h each time.</li>
	<li>For easier visualization of cells under fluorescent microscopy, stain cells with DAPI during the third of the five washes. Dilute DAPI stock concentration (5 mg/mL, stored at 4 &#176;C) in 1:5,000 dilutions in wash water. Incubate the gel with a DAPI-water solution (2 mL) for 30 min to 1 h.</li>
	<li>Wash two additional times with 3 mL of water.</li>
	<li>Expand gels in any flat dish that is big enough to contain the samples. The expanded gels which are prepared on 0.4 cm2 area coverglass fit nicely in a glass bottom 6-well plate. 
	NOTE: Post-expansion-stained samples can be stored for up to 4 months in a PBS buffer at 4&#176; C. Add enough water to avoid drying and protect the sample from photobleaching. Repeat the expansion and DAPI staining step before imaging. To avoid any risk of fluorophore degradation, samples need to be imaged as soon as possible.</li>
</ol>
11. Imaging
<ol>
	<li>Coat the 6-well glass bottom imaging chamber with 0.01% poly-L-lysine (3 mL each) to immobilize the gel samples.</li>
	<li>Transfer gel samples to the coated imaging chamber.</li>
	<li>Image the samples with any desired fluorescence scopes.</li>
</ol>

<ol>
	<li>Shi, X., 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=Label-retention+expansion+microscopy.">Label-retention expansion microscopy.</a> The Journal of Cell Biology. 220 (9), 202105067 (2021).</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>Truckenbrodt, S., Sommer, C., Rizzoli, S. O., Danzl, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+optimization+in+X10+expansion+microscopy.">A practical guide to optimization in X10 expansion microscopy.</a> Nature Protocols. 14 (3), 832-863 (2019).</li><li>Wang, Y., 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=Combined+expansion+microscopy+with+structured+illumination+microscopy+for+analyzing+protein+complexes.">Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes.</a> Nature Protocols. 13 (8), 1869-1895 (2018).</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>Gautier, 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=An+engineered+protein+tag+for+multiprotein+labeling+in+living+cells.">An engineered protein tag for multiprotein labeling in living cells.</a> Chemistry &amp; Biology. 15 (2), 128-136 (2008).</li><li>Keppler, A., Pick, H., Arrivoli, C., Vogel, H., Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+fusion+proteins+with+synthetic+fluorophores+in+live+cells.">Labeling of fusion proteins with synthetic fluorophores in live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (27), 9955-9959 (2004).</li><li>Thevathasan, J. V., 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=Nuclear+pores+as+versatile+reference+standards+for+quantitative+super+resolution+microscopy.">Nuclear pores as versatile reference standards for quantitative super resolution microscopy.</a> Nature Methods. 16 (10), 1045-1053 (2019).</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 (19), 45836 (2018).</li><li>Damstra, H. G. 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=Visualizing+cellular+and+tissue+ultrastructure+using+Ten-fold+Robust+Expansion+Microscopy+(TREx).">Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).</a> eLife. 11, 73775 (2021).</li><li>M'Saad, O., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+of+proteins+in+their+ultrastructural+context.">Light microscopy of proteins in their ultrastructural context.</a> Nature Communications. 11 (1), 3850 (2020).</li></ol>

The authors declare no conflicts of interest.

The key innovation of LR-ExM is to use trifunctional anchors to effectively label the target proteins and improve image quality. This method is limited by trifunctional anchors, which are not so readily available to researchers. However,&#160;trifunctional anchors can be shared with other researchers upon request, and similar products such as ExM probes from Chrometa are now commercially available as well.
In this protocol, 1 h incubation at room temperature has been performed for the primary ...

Expansion microscopy (ExM) has been used by researchers since it was first introduced as a convenient approach to achieve super resolution imaging with conventional microscopes, such as epifluorescence and confocal microscopes1,2,3,4,5,6,7. Using ExM, it is possible to achieve ~70 nm lateral resolution even with regular confocal microscopes. When ExM is combined with super-resolution imaging, the resolution is further improved. For instance, one can achieve roughly 30 nm resolution with structured illumination microscopy (SIM), and roughly 4 nm resolution with stochastic optical reconstruction microscopy (STORM)1,5.
However, low labeling efficiency is a critical issue with standard ExM methods. Fluorescence loss can vary based on the type of fluorescent groups and digestion time. On average, however, it has been reported that more than 50% of fluorophores are lost after the polymerization and the protein digestion steps of ExM, which is detrimental to the imaging quality3,4.
Thus, label-retention expansion microscopy (LR-ExM) has been developed, which can efficiently retain labels and reduce signal loss1. The key innovation of LR-ExM is the use of a set of trifunctional anchors instead of merely using fluorescent dyes-as in the standard ExM procedure-for staining proteins of interest. These trifunctional linkers consist of three parts: (1) the connector (e.g., N-hydroxysuccinimide (NHS)) to connect to the antibody, (2) the anchor (e.g., methacrylamide (MA)) to anchor proteins to the polymer, and (3) the reporter (e.g., biotin or digoxigenin (DIG)) to conjugate to an organic dye. The trifunctional anchors survive the polymerization and protein digestion steps, and therefore prevent fluorophore loss.
Furthermore, this method holds great potential since it is compatible with self-labeling enzymatic tags such as SNAP or CLIP. Enzymatic tag approaches have some benefits over the immunostaining approach regarding high specificity and labeling efficiency8,9,10.
In this manuscript, a detailed procedure of LR-ExM is demonstrated. LR-ExM is a highly effective and flexible method to achieve high spatial resolution with enhanced labeling efficiency.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylamide&#160;</td>
			<td>Sigma&#160;</td>
			<td>A9099&#160;</td>
			<td>ExM Gel</td>
		</tr>
		<tr>
			<td>AffiniPure Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>H+L, 711&#8211;005-152</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>AffiniPure Donkey Anti-Rat IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>H+L, 712&#8211;005-153</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Streptavidin</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-540-084</td>
			<td>Fluorescent probes&#160;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Streptavidin</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-580-084</td>
			<td>Fluorescent probes&#160;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Streptavidin</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-600-084</td>
			<td>Fluorescent probes&#160;</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate&#160;</td>
			<td>Sigma&#160;</td>
			<td>A3678&#160;</td>
			<td>ExM Gel</td>
		</tr>
		<tr>
			<td>anti-H3K4me3</td>
			<td>Abcam</td>
			<td>ab8580</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>anti-H3K9me3</td>
			<td>Abcam</td>
			<td>ab176916</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>DAPI dilacetate</td>
			<td>Thermofisher Scientific</td>
			<td>D3571</td>
			<td>Fluorescent probes&#160;</td>
		</tr>
		<tr>
			<td>DyLight 488 Labeled Anti-Digoxigenin/Digoxin (DIG)</td>
			<td>Vector Laboratories</td>
			<td>DI-7488</td>
			<td>Fluorescent probes&#160;</td>
		</tr>
		<tr>
			<td>DyLight 594 Labeled Anti-Digoxigenin/Digoxin (DIG)</td>
			<td>Vector Laboratories</td>
			<td>DI-7594</td>
			<td>Fluorescent probes&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>EMD Millipore Corp.</td>
			<td>324626-25GM</td>
			<td>Fixation buffer</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma&#160;</td>
			<td>EDTA</td>
			<td>Digestion buffer</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 10% EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>50-262-13</td>
			<td>Anchoring</td>
		</tr>
		<tr>
			<td>Grace Bio-Labs CultureWell removable chambered coverglass</td>
			<td>Grace Bio-Labs&#160;</td>
			<td>GBL112358-8EA</td>
			<td>Cell culture chamber</td>
		</tr>
		<tr>
			<td>Grace Bio-Labs CultureWell removal tool</td>
			<td>Grace Bio-Labs&#160;</td>
			<td>GBL103259</td>
			<td>Removal tool</td>
		</tr>
		<tr>
			<td>Guanidine HCl&#160;</td>
			<td>Sigma&#160;</td>
			<td>G3272&#160;</td>
			<td>Digestion buffer</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266-1KG</td>
			<td>Fixation buffer</td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5a</td>
			<td>ATCC</td>
			<td>30&#8211;2007</td>
			<td>Celll culture medium</td>
		</tr>
		<tr>
			<td>Methacrylic acid N-hydroxysuccinimide ester,98%&#160; (MA-NHS)</td>
			<td>Sigma&#160;</td>
			<td>730300-1G</td>
			<td>Anchoring</td>
		</tr>
		<tr>
			<td>monoclonal mouse anti-Nup153 antibody</td>
			<td>Abcam</td>
			<td>ab24700</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>N,N&#8242;Methylenebisacrylamide&#160;</td>
			<td>Sigma&#160;</td>
			<td>M7279&#160;</td>
			<td>ExM Gel</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242; Tetramethylethylenediamine&#160;(TEMED)</td>
			<td>Sigma&#160;</td>
			<td>T7024&#160;</td>
			<td>ExM Gel</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Aqueous Solutions</td>
			<td>Electron Microscopy Sciences</td>
			<td>50-980-487</td>
			<td>Fixation buffer</td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757-25G</td>
			<td>Fixation buffer</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P8920-100ML</td>
			<td>Chamber coating</td>
		</tr>
		<tr>
			<td>Proteinase K&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P4850-5ML</td>
			<td>Digestion buffer</td>
		</tr>
		<tr>
			<td>Rabbit anti-clathrin heavy-chain antibody</td>
			<td>Abcam</td>
			<td>ab21679</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>rat anti&#8211;&#945;-tubulin antibody,tyrosinated, clone YL1/2</td>
			<td>Millipore Sigma</td>
			<td>MAB1864-I</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Sodium Acrylate&#160;</td>
			<td>Sigma</td>
			<td>408220</td>
			<td>ExM Gel</td>
		</tr>
		<tr>
			<td>Streptavidin / Biotin blocking kit</td>
			<td>Vector Laboratories</td>
			<td>SP-2002</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Tris-HCl&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>AM9855&#160;</td>
			<td>Digestion buffer</td>
		</tr>
		<tr>
			<td>U2OS</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>6 well glass bottom plates</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td>Imaging plate</td>
		</tr>
	</tbody>
</table>

label-retention expansion microscopy (lr-exm) enables super-resolution imaging and high-efficiency labeling

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After embedding cells or tissues in swellable hydrogel, samples can be physically expanded three-to sixteen-fold (linear dimension) compared to the original size. Therefore, the effective resolution of any microscope is increased by the expansion factor. A major limitation of the previously introduced ExM is reduced fluorescence after polymerization and the digestion procedure. To overcome this limitation, label-retention expansion microscopy (LR-ExM) has been developed, which prevents signal loss and greatly enhances labeling efficiency using a set of novel trifunctional anchors. This technique allows one to achieve higher resolution when investigating cellular or subcellular structures at a nanometric scale with minimal fluorescent signal loss. LR-ExM can be used not only for immunofluorescence labeling, but also with self-labeling protein tags, such as SNAP- and CLIP-tags, thus achieving higher labeling efficiency. This work presents the procedure and troubleshooting for this immunostaining-based approach, as well as discussion of self-labeling tagging approaches of LR-ExM as an alternative.

Clathrin-coated pits (CCPs) are immunostained using trifunctional anchors (Figure 1B) and LR-ExM is performed as described in Figure 1A. LR-ExM (Figure 2C,E) shows much higher fluorescence intensity compared to the protein-retention expansion microscopy&#160;(proExM, Figure 2A) or biotin-ExM (Figure 2B); the signal for LR-ExM was about six times higher than...

Watch this Scientific Journal Video about Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling at JoVE.com

Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling

A protocol of label retention expansion microscopy (LR-ExM) is demonstrated. LR-ExM uses a novel set of trifunctional anchors, which provides better labeling efficiency compared to previously introduced expansion microscopies.

Label-Retention Expansion Microscopy (LR-ExM) Enables Super-Resolution Imaging and High-Efficiency Labeling

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After ...

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3.5K Views.  University of California Irvine. A protocol of label retention expansion microscopy (LR-ExM) is demonstrated. LR-ExM uses a novel set of trifunctional anchors, which provides better labeling efficiency compared to previously introduced expansion microscopies.

Video: Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...