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><tbody><tr><td>Acrylamide&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A9099&amp;nbsp;</td><td>ExM Gel</td></tr><tr><td>AffiniPure Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>H+L, 711&amp;ndash;005-152</td><td>Antibody</td></tr><tr><td>AffiniPure Donkey Anti-Rat IgG</td><td>Jackson ImmunoResearch</td><td>H+L, 712&amp;ndash;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&amp;nbsp;</td></tr><tr><td>Alexa Fluor 594 Streptavidin</td><td>Jackson ImmunoResearch</td><td>016-580-084</td><td>Fluorescent probes&amp;nbsp;</td></tr><tr><td>Alexa Fluor 647 Streptavidin</td><td>Jackson ImmunoResearch</td><td>016-600-084</td><td>Fluorescent probes&amp;nbsp;</td></tr><tr><td>Ammonium Persulfate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A3678&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>DyLight 488 Labeled Anti-Digoxigenin/Digoxin (DIG)</td><td>Vector Laboratories</td><td>DI-7488</td><td>Fluorescent probes&amp;nbsp;</td></tr><tr><td>DyLight 594 Labeled Anti-Digoxigenin/Digoxin (DIG)</td><td>Vector Laboratories</td><td>DI-7594</td><td>Fluorescent probes&amp;nbsp;</td></tr><tr><td>EGTA</td><td>EMD Millipore Corp.</td><td>324626-25GM</td><td>Fixation buffer</td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma&amp;nbsp;</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&amp;nbsp;</td><td>GBL112358-8EA</td><td>Cell culture chamber</td></tr><tr><td>Grace Bio-Labs CultureWell removal tool</td><td>Grace Bio-Labs&amp;nbsp;</td><td>GBL103259</td><td>Removal tool</td></tr><tr><td>Guanidine HCl&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>G3272&amp;nbsp;</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&#039;s 5a</td><td>ATCC</td><td>30&amp;ndash;2007</td><td>Celll culture medium</td></tr><tr><td>Methacrylic acid N-hydroxysuccinimide ester,98%&amp;nbsp; (MA-NHS)</td><td>Sigma&amp;nbsp;</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&amp;prime;Methylenebisacrylamide&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>M7279&amp;nbsp;</td><td>ExM Gel</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime; Tetramethylethylenediamine&amp;nbsp;(TEMED)</td><td>Sigma&amp;nbsp;</td><td>T7024&amp;nbsp;</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&amp;nbsp;</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&amp;ndash;&amp;alpha;-tubulin antibody,tyrosinated, clone YL1/2</td><td>Millipore Sigma</td><td>MAB1864-I</td><td>Antibody</td></tr><tr><td>Sodium Acrylate&amp;nbsp;</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&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>AM9855&amp;nbsp;</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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Biology

3.6K 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

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Biochemistry

Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. Conversely, the requirement of fluorophore labeling in fluorescent techniques poses significant challenges, such as photobleaching and non-uniform labeling of fluorescent probes and prolonged sample processing. In this protocol, the detailed working procedures of vibrational imaging of swelled tissue and analysis (VISTA) are presented. VISTA circumvents obstacles associated with fluorophores and achieves label-free super-resolution volumetric imaging in biological samples with spatial resolution down to 78 nm. The procedure is established by embedding cells and tissues in hydrogel, isotropically expanding the hydrogel sample hybrid, and visualizing endogenous protein distributions by vibrational imaging with stimulated Raman scattering microscopy. The method is demonstrated on both cells and mouse brain tissues. Highly correlative VISTA and immunofluorescence images were observed, validating the protein origin of imaging specificities. Exploiting such correlation, a machine learning-based image-segmentation algorithm was trained to achieve multi-component prediction of nuclei, blood vessels, neuronal cells, and dendrites from label-free mouse brain images. The procedure was further adapted to investigate pathological poly-glutamine (polyQ) aggregates in cells and amyloid-beta (A&#946;) plaques in brain tissues with high throughput, justifying its potential for large-scale clinical samples.

By combining sample-expansion hydrogel chemistry with label-free chemical-specific stimulated Raman scattering microscopy, the protocol describes how to achieve label-free super-resolution volumetric imaging in biological samples. With an additional machine learning image segmentation algorithm, protein-specific multi-component images in tissues without antibody labeling were obtained.

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. ...

Chemistry

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

Here, a protocol for the implementation of expansion microscopy in early Drosophila embryos to achieve super-resolution imaging using a conventional laser-scanning confocal microscope is presented.

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged ...

Developmental Biology

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

Neuroscience

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 ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

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 ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

Expansion Microscopy for Robust 11-Fold Expansion

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  

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 ...

Expansion Microscopy for Visualizing Intracellular Sialylated Glycans via MOE

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

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

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Chapters in this video

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

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

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Universal Molecular Retention with 11-Fold Expansion Microscopy

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy