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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 microscopes^{1,2,3,4,5,6,7}. 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 quality^3,4.

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 efficiency^8,9,10.

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.

Protokół

1. Cell culture

1. Use U2OS cells cultured in McCoy's 5A medium supplemented with 10% FBS at 37 °C in 5% CO₂.
2. Culture cells onto a 16 well removable chambered coverglass (culture area 0.4 cm²) for ease of handling.

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).

1. Once cell counts reach ~0.04 x 10⁶, fix cells with 100 µL 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.
2. Wash cells three times for 5 min each with 200 µL of phosphate-buffered saline (PBS).
3. Permeabilize cells with the 100 µL of permeabilization buffer at room temperature for 15 min (Table 1).
   ​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 °C. Replace any evaporated PBS and protect the sample from photobleaching.

3. Endogenous streptavidin and biotin blocking

1. Incubate cells with the streptavidin solution diluted in a cell blocking buffer (100 µL) for 15 min at room temperature (Table 1).
2. Rinse briefly with 200 µL of cell blocking buffer.
3. Incubate cells with 100 µL of biotin solution diluted in a cell blocking buffer for 15 min at room temperature.
   ​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.

4. Primary antibody staining

1. Incubate cells with rat anti-α-tubulin antibody (1:500 dilution) and rabbit anti-clathrin heavy-chain antibody (1:100 dilution) in 100 µL of cell blocking buffer for 16 h at 4 °C or 1 h at room temperature. Double the incubation time for the tissue samples.
2. Wash samples four times with 200 µL of PBS for 5 min each.

5. LR-ExM specific secondary antibody staining

1. 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.
2. 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 µL of cell blocking buffer for 1 h at room temperature. Double this incubation time for the tissue samples.
3. Wash samples four times in 200 µL of PBS for 5 min each.

6. Additional anchoring

1. Incubate cells with 0.25% glutaraldehyde (GA) in PBS (100 µL) 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.
2. Wash samples three times in PBS for 5 min each.

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.

1. Remove the upper structure of the 16-well gel plate. Add 40 µL of monomer solution to each well to condition cells on ice. Incubate on ice for 5 min.
   1. To prepare a monomer solution, see Table 2.
2. Prepare the gelation solution on ice. Add double distilled water and 10% N,N,N′,N′ Tetramethylethylenediamine (TEMED) accelerator to the monomer solution (10% TEMED: monomer solution = 1:47); do not add the initiator yet (Table 3).
3. For a cell culture chamber with an area of 0.4 cm², use a gelation solution volume of about 40 µL. Prepare 45 µL of gelation solution for each well.
4. Add 10% ammonium persulfate (APS) to the gelation solution, incubate on ice, and immediately pipette 40 µL 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).
5. Protect the sample from light and move the coverglass in the 10 cm Petri dish to a 37 °C incubator for 1.5 h for gelation. To keep the humidity of the gel during the 37 °C incubation, cover the Petri dish with the lid, and put a few drops of water in the Petri dish.

8. Digestion

1. 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 °C. Use at least 10-fold excess volume of digestion buffer.
2. 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.
   ​NOTE: Gel samples can be stored for up to 1 month in a PBS buffer at 4 °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.

9. Post-digestion fluorescence staining

1. Incubate gels in 2 mL volume of streptavidin (STV)/digoxigenin (DIG) staining buffer with 2 to 5 µM STV-dye and/or anti-DIG-dye for 24 h at room temperature. Keep the samples in the dark.

10. Expansion

1. Wash and expand the gel four times with excessive water (2-3 mL) for 30 min to 1 h each time.
2. 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 °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.
3. Wash two additional times with 3 mL of water.
4. 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° 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.

11. Imaging

1. Coat the 6-well glass bottom imaging chamber with 0.01% poly-L-lysine (3 mL each) to immobilize the gel samples.
2. Transfer gel samples to the coated imaging chamber.
3. Image the samples with any desired fluorescence scopes.

Wyniki

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 (proExM, Figure 2A) or biotin-ExM (Figure 2B); the signal for LR-ExM was about six times higher than...

Dyskusje

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, 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 ...

Materiały

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