Expansion microscopy of C. elegans
- Department of Biological Engineering, Massachusetts Institute of Technology, United States
- Media Lab, Massachusetts Institute of Technology, United States
- McGovern Institute, Massachusetts Institute of Technology, United States
- Division of Health Sciences and Technology, Massachusetts Institute of Technology, United States
- Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, United States
- Broad Institute of MIT and Harvard, United States
- Department of Molecular and Cellular Physiology, Stanford University, United States
- Federated Department of Biological Sciences, New Jersey Institute of Technology and Rutgers University-Newark, United States
- The Brain Research Institute, New Jersey Institute of Technology, United States
- Koch Institute, Massachusetts Institute of Technology, United States
- Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, United States
Figures
Figure 1
Workflow for expansion of C. elegans (ExCel) sample processing.
A method for expanding cuticle-enclosed intact C. elegans, extending published proExM and ExFISH protocols with specific modifications (shown in green text; full key in lower left). Depending on …
Figure 2
ExCel enables antibody-mediated visualization of fluorescent proteins.
(A) Representative images of immunohistochemistry against GFP in paraformaldehyde-fixed, β-mercaptoethanol-reduced (as in Figure 1A–C) hermaphrodite animals, on which the antibody staining was …
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Figure 2—source data 1
Intensity measurements for the signal-to-background ratios shown in Figure 2D.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig2-data1-v1.mat
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Figure 2—source data 2
Intensity measurements for the signal-to-background ratios shown in Figure 2F.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig2-data2-v1.mat
Figure 3 with 2 supplements
Isotropy of ExCel.
(A) Representative images of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G) hermaphrodite animals in the second larval stage (‘Larval …
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Figure 3—source data 1
Root-mean-square (RMS) length measurement errors plotted in Figure 3B.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig3-data1-v1.mat
Figure 3—figure supplement 1
Local distortion at the gonad region of day 1 – day 2 adult hermaphrodites.
Representative images of adult gonad regions of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G) hermaphrodite animals before Proteinase …
Figure 3—figure supplement 2
Local distortion at the mouth region.
Representative images of mouth regions of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded L1 – day 2 adult hermaphrodite C. elegans animals (as in Figure 1A–C, …
Figure 4
Post-ExCel NHS-ester staining reveals anatomical structures.
Representative images of (A) pharyngeal region, (B) intestinal tissue and (C) gonad tissue of ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, …
Figure 5
ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical features.
The pharyngeal region of a representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figu…
Figure 6
Super-resolution imaging of synaptic proteins with ExCel.
(A) Representative images of GFP-fused synaptic proteins RAB-3, SNB-1 and GLR-1 in paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G) …
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Figure 6—source data 1
Line intensity profiles for all data points plotted in Figure 6C.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig6-data1-v1.mat
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Figure 6—source data 2
Peak counts for all data points plotted in Figure 6C.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig6-data2-v1.xlsx
Figure 7
Super-resolution imaging of electrical synapses with ExCel.
Representative images of TagRFP-fused innexin protein CHE-7 in a paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G) L4 hermaphrodite …
Figure 8
RNA detection in neurons.
ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N) hermaphrodite animals …
Figure 9
RNA detection in neurons, at sub-cellular resolution.
Representative images of ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N) …
Figure 10
Single-neuron resolution RNA quantification.
(A) A representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as in Figure 1A–I, M, N) L4 …
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Figure 10—source data 1
Count of unc-25, cat-2 and tph-1 RNA molecules within single neurons, whose population statistics are shown in Figure 10D-F.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig10-data1-v1.xlsx
Figure 11
Screen of post-gelation treatments that confer tissue expandability and general stainability of epitopes.
(A) Representative transillumination images of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded hermaphrodite animals, (left column) right after hydrogel …
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Figure 11—source data 1
Measurements for the expandabiliy and stainability assays, whose population statistics are summarized in Figure 11C.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig11-data1-v1.mat
Figure 12
Immunohistochemistry after selected post-gelation treatments.
(A) Estimated signal-to-noise ratio of immunostaining in paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded hermaphrodite animals, where the immunostaining was …
Figure 13 with 1 supplement
Workflow for epitope-preserving expansion of C. elegans (epitope-preserving ExCel) sample processing.
A method for expanding cuticle-enclosed intact C. elegans, while permitting readout of a majority of antigens that are detectable through non-IgM-class antibodies (~70%; estimated from the …
Figure 13—figure supplement 1
DAPI staining after epitope-preserving ExCel.
Representative images of epitope-preserving-ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, collagenase-digested, denaturation-processed, …
Figure 14
Isotropy of epitope-preserving ExCel.
(A) Representative images of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded (as in Figure 1A–C, E–G) hermaphrodite animals in the second larval stage (‘Larval …
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Figure 14—source data 1
Root-mean-square (RMS) length measurement errors plotted in Figure 14B.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig14-data1-v1.mat
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Figure 14—source data 2
Root-mean-square (RMS) length measurement errors plotted in Figure 14D.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig14-data2-v1.mat
Figure 15
Epitope-preserving ExCel allows multiplexed imaging of endogenous proteins at nanoscale resolution.
A representative epitope-preserving-ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, collagenase-digested, denaturation-processed, antibody-stained, …
Figure 16
Super-resolution imaging of pre-synaptic active and peri-active zones with epitope-preserving ExCel.
A representative epitope-preserving-ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, collagenase-digested, denaturation-processed, antibody-stained, …
Figure 17
Workflow for iterative expansion of C. elegans (iExCel) sample processing.
A method for iteratively expanding cuticle-enclosed intact C. elegans, for a final linear expansion factor of ~20x. Sample processing prior to Panel A is identical to the workflow for the standard …
Figure 18 with 1 supplement
iExCel-expanded whole C. elegans.
A representative iExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, hydrogel-embedded, Proteinase-K digested, antibody-stained, second-gel-re-embedded, linker-hybridized, …
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Figure 18—source data 1
Root-mean-square (RMS) length measurement errors plotted in Figure 18—figure supplement 1.
- https://cdn.elifesciences.org/articles/46249/elife-46249-fig18-data1-v1.mat
Figure 18—figure supplement 1
Isotropy of iExCel.
Root-mean-square length measurement error (‘RMS Error’) computed from pre-expansion images (as acquired in the left panel of Figure 18A) and post-2nd-round-expansion images (as acquired in the right …
Appendix 1—figure 1
Construction of the gelling chamber.
Videos
Video 1
Post-ExCel NHS-ester staining of the pharyngeal region.
Confocal stack of the pharyngeal region of the L3 hermaphrodite animal shown in Figure 4A. Scale bar: 20 μm.
Video 2
Post-ExCel NHS-ester staining of the gut and germline tissue.
Confocal stack of the upper body region (between the pharynx and the vulva) of the L4 hermaphrodite animal shown in Figure 4B–C. Scale bar: 20 μm.
Video 3
ExCel 4-color readout.
Full confocal stack of the L2 hermaphrodite shown in Figure 5. Scale bars: 10 μm.
Video 4
Sub-cellular localization of unc-25 transcripts in motor neurons.
The retrovesicular ganglion of a representative ExCel-processed (formaldehyde-fixed, β-mercaptoethanol-reduced, LabelX- and AcX-treated, hydrogel-embedded, Proteinase-K digested and re-embedded; as …
Video 5
iExCel-expanded nerve ring.
Full confocal stack of the pharyngeal region of the 2nd-round-expanded L3 hermaphrodite shown in the right panel of Figure 18B. Scale bar: 10 μm.
Tables
Table 1
Screened post-gelation treatments.
Experimental parameters used for each post-hydrogel-embedding treatment that was screened and shown in Figure 11C.
| Treatment type | Treatment name | Treatment buffer | Buffer pH | Protease concentration | Duration (triple numbers, times at 37oC-70oC-95oC, in hr) |
|---|---|---|---|---|---|
| No treatment | No treatment | TNT (Tris, 1M NaCl, Triton) | 8.0 | N/A | 0-0-0 (kept at RT) |
| Proteinase K | Proteinase K (standard ExCel) | 50 mM Tris pH 8 + 0.5M NaCl + 40 mM CaCl2 + 0.1% Triton | 8.0 | 8 U/mL | 2 days 37oC* |
| Non-Proteinase-K proteases | Trypsin (standard) | 50 mM Tris pH 8 + 0.5M NaCl + 40 mM CaCl2 | 8.0 | 1 mg/mL | 5 days 37oC* |
| Trypsin (seq grade) | 50 mM Tris pH 8 + 0.5M NaCl + 40 mM CaCl2 | 8.0 | 0.1 mg/mL | 5 days 37oC** | |
| Elastase | 50 mM Tris pH 9 + 0.5M NaCl + 40 mM CaCl2 | 9.0 | 0.5 mg/mL | 5 days 37oC* | |
| Pepsin | 3 mM HCl + 0.5M NaCl + 40 mM CaCl2 | 2.5 | 1 mg/mL | 5 days 37oC* | |
| Thermolysin | 50 mM Tris pH 8 + 0.5M NaCl + 40 mM CaCl2 | 8.0 | 0.5 mg/mL | 5 days 70oC* | |
| Papain | 1x PBS pH 6.5 + 5 mM L-cysteine + 5 mM EDTA + 2M NaCl | 6.5 | 1 mg/mL | 5 days 70oC* | |
| Alpha-chymotrypsin | 50 mM Tris pH 8 + 0.5M NaCl + 40 mM CaCl2 | 8.0 | 1 mg/mL | 5 days 25oC* | |
| Collagenase Type II | 50 mM Tris pH 8 + 0.5M NaCl + 40 mM CaCl2 | 8.0 | 1 U/mL | 5 days 37oC* | |
| Antigen retrieval | EDTA pH 8 | 10 mM Tris + 1 mM EDTA + 2M NaCl | 8.0 | N/A | 18-18-2 |
| EDTA-Tris pH 9 | 50 mM Tris + 1 mM EDTA + 0.05% Tween + 2M NaCl | 9.0 | N/A | 18-18-2 | |
| Citrate pH 6 | 10 mM citrate pH 6 + 0.05% Tween + 2M NaCl | 6.0 | N/A | 18-18-2 | |
| VC low | 5% (w/v) ascorbic acid + 2M NaCl | 3.0 | N/A | 1-1-1 | |
| VC high | 5% (w/v) ascorbic acid + 2M NaCl | 3.0 | N/A | 3-24-2 | |
| Heat denaturation in MAP-based buffers | MAP1 18-18-2 | 50 mM Tris + 200 mM SDS + 200 mM NaCl | 9.0 | N/A | 18-18-2 |
| MAP5 18-18-2 | 50 mM Tris + 200 mM SDS + 400 mM NaCl + 20 mM CaCl2 | 9.0 | N/A | 18-18-2 | |
| MAP5 120-18-2 | 50 mM Tris + 200 mM SDS + 400 mM NaCl + 20 mM CaCl2 | 9.0 | N/A | 120-18-2 | |
| MAP5 18-120-2 | 50 mM Tris + 200 mM SDS + 400 mM NaCl + 20 mM CaCl2 | 9.0 | N/A | 18-120-2 | |
| MAP5 18-18-120 | 50 mM Tris + 200 mM SDS + 400 mM NaCl + 20 mM CaCl2 | 9.0 | N/A | 18-18-120 | |
| MAP5 18-18-18 | 50 mM Tris + 200 mM SDS + 400 mM NaCl + 20 mM CaCl2 | 9.0 | N/A | 18-18-18 | |
| MAP5 18-18-2-2 | 50 mM Tris + 200 mM SDS + 400 mM NaCl + 20 mM CaCl2 | 9.0 | N/A | 18-18-2, and 2 hr at 121oC |
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* Multi-day protease treatments are refreshed with newly prepared solutions every day, to partially compensate for loss of enzyme activity over time.
** Refreshment for Trypsin (seq grade) was performed only on Day 1, 3, 5, instead of daily, due to limits on reagent availability.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Caenor-habditis elegans) | N2 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00000001 | Genotype: wild-type |
| Strain, strain background (C. elegans) | CX16682 | This paper | Genotype: kyIs700 [tag-168p::GFP; tag-168p::rpl-22-3xHA] ? (‘?” denotes that the chromosome where the construct got integrated is unknown) | |
| Strain, strain background (C. elegans) | CZ1632 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00005366 | Genotype: juIs76 [unc-25p::GFP + lin-15(+)] II |
| Strain, strain background (C. elegans) | NM2415 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00029065 | Genotype: jsIs682 [rab-3p::GFP::rab-3 + lin-15(+)] III |
| Strain, strain background (C. elegans) | KP1148 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00023626 | Genotype: nuIs25 [glr-1p::glr-1::GFP + lin-15(+)] ? |
| Strain, strain background (C. elegans) | CZ333 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00005345 | Genotype: juIs1 [unc-25p::snb-1::GFP + lin-15(+)] IV |
| Strain, strain background (C. elegans) | CF702 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00004831 | Genotype: muIs32 [mec-7p: :GFP + lin-15(+)] II |
| Strain, strain background (C. elegans) | NQ570 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00029098 | Genotype: qnIs303 [hsp-16.2p::flp-13 + hsp-16.2p::GFP + rab-3p::mCherry] IV |
| Strain, strain background (C. elegans) | AML32 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00000192 | Genotype: wtfIs5 [rab-3p::NLS::GCaMP6s + rab-3p::NLS::TagRFP] ? |
| Strain, strain background (C. elegans) | OH16372 | This paper | Genotype: che-7(ot866[che-7::TagRFP)] | |
| Antibody | anti-GFP (chicken polyclonal) | Abcam | Cat#: ab13970; RRID:AB_300798 | IHC (1:100) |
| Antibody | anti-GFP (rabbit polyclonal) | Thermo Fisher Scientific | Cat#: A11122; RRID:AB_221569 | IHC (1:200) |
| Antibody | anti-mCherry (rabbit polyclonal) | Kerafast | Cat#: EMU106 | IHC (1:500 – 1:2000) |
| Antibody | anti-TagRFP (guinea pig polyclonal) | Kerafast | Cat#: EMU108 | IHC (1:500 –1:2000) |
| Antibody | anti-RFP (rabbit polyclonal) | Thermo Fisher Scientific | Cat#: R10367; RRID:AB_2315269 | IHC (1:100) |
| Antibody | anti-DLG-1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: DLG1; RRID:AB_2314321 | IHC (5 μg/mL) |
| Antibody | anti-UNC-10 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: RIM; RRID:AB_579790 | IHC (5 μg/mL) |
| Antibody | anti-UNC-10 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: RIM2; RRID:AB_10570332 | IHC (5 μg/mL) |
| Antibody | anti-myotactin (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: MH46; RRID:AB_528387 | IHC (5 μg/mL) |
| Antibody | anti-SAX-7 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: CeSAX7; RRID:AB_10567266 | IHC (5 μg/mL) |
| Antibody | anti-SNB-1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: SB1; RRID:AB_579792 | IHC (5 μg/mL) |
| Antibody | anti-DYN-1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: DYN1; RRID:AB_10572297 | IHC (5 μg/mL) |
| Antibody | anti-acetylated-tubulin (mouse monoclonal) | Millipore Sigma | Cat#: T7451; RRID:AB_609894 | IHC (5 μg/mL) |
| Antibody | anti-DAO-5 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: DAO5; RRID:AB_10573805 | IHC (5 μg/mL) |
| Antibody | anti-LMP-1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: LMP1; RRID:AB_2161795 | IHC (5 μg/mL) |
| Antibody | anti-LMN-1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: LMN1; RRID:AB_10573809 | IHC (5 μg/mL) |
| Antibody | anti-HCP-4 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: HCP4; RRID:AB_2078913 | IHC (5 μg/mL) |
| Antibody | anti-CYP-33E1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: CYP33E1; RRID:AB_10571938 | IHC (5 μg/mL) |
| Antibody | anti-HMR-1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: HMR1; RRID:AB_2153752 | IHC (5 μg/mL) |
| Antibody | anti-LET-413 (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#: LET413; RRID:AB_10571452 | IHC (5 μg/mL) |
| Antibody | anti-chicken-IgY (donkey polyclonal) | Jackson Immuno-Research | Cat#: 703-005-155; RRID:AB_2340346 | IHC (10 μg/mL) |
