1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
|
# Expansion microscopy
The core intuition of expansion microscopy is that nanoscale information can be preserved topologically in the polymer network and revealed by uniform metric expansion rather than by improved optics. Expansion microscopy (ExM) exploits polymer physics to physically magnify otherwise sub-diffraction biological ultrastructure by covalently linking biomolecular targets (or their labels) to a highly water-swellable, charge-dense hydrogel, then homogeneously dilating that hydrogel to change its size.
* Hümpfer & Sauer, 2024 — *A cell biologist’s guide to ExM*. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11058692/?utm_source=chatgpt.com)
* Gao & Boyden, 2017 — *Q\&A: Expansion microscopy*. [BioMed Central](https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-017-0393-3?utm_source=chatgpt.com)
* Behzadi et al., 2024/2025 — *ExM in genetic animal models (review)*. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11660448/?utm_source=chatgpt.com), [arXiv](https://arxiv.org/abs/2411.06676?utm_source=chatgpt.com)
* Louvel et al., 2023 — *iU-ExM*. [Nature](https://www.nature.com/articles/s41467-023-43582-8?utm_source=chatgpt.com)
* Freifeld et al., 2017 — Zebrafish ExM. [PNAS](https://www.pnas.org/doi/10.1073/pnas.1706281114?utm_source=chatgpt.com)
* Yu et al., 2020 — C. elegans ExM (ExCel). [eLife](https://elifesciences.org/articles/46249?utm_source=chatgpt.com)
* **ExSeq (Science, 2021)** — expansion microscopy + in situ sequencing gives nanoscale-resolved RNA maps. [Science](https://www.science.org/doi/10.1126/science.aax2656?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7900882/?utm_source=chatgpt.com)
[One-step nanoscale expansion microscopy reveals individual protein shapes](https://www.nature.com/articles/s41587-024-02431-9) with comparisons to the predicted shapes from alphafold.
## Core, foundational ExM papers
* **Original expansion microscopy paper** — Chen *et al.*, *Science* (2015). First description of embedding, anchoring, homogenization, and swelling. [Science](https://www.science.org/doi/10.1126/science.1260088?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4312537/?utm_source=chatgpt.com)
* **proExM (protein-retention)** — Tillberg *et al.*, *Nat. Biotechnol.* (2016). Anchors proteins directly to the gel; compatible with antibodies/FPs. [PubMed](https://pubmed.ncbi.nlm.nih.gov/27376584/?utm_source=chatgpt.com)
* **MAP (Magnified Analysis of the Proteome)** — Ku *et al.*, *Nat. Biotechnol.* (2016). 4× expansion of intact tissues with proteome preservation. [PubMed](https://pubmed.ncbi.nlm.nih.gov/27454740/?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5070610/?utm_source=chatgpt.com)
* **iExM (iterative ExM)** — Chang *et al.*, *Nat. Methods* (2017). Serial expansions toward \~20×. [Nature](https://www.nature.com/articles/nmeth.4261?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/28417997/?utm_source=chatgpt.com)
## Widely used variants & protocols
* **U-ExM (ultrastructure ExM)** — Gambarotto *et al.*, *Nat. Methods* (2019) and protocol follow-ups. Preserves ultrastructure for EM-like context. [PubMed](https://pubmed.ncbi.nlm.nih.gov/30559430/?utm_source=chatgpt.com), [ScienceDirect](https://www.sciencedirect.com/science/article/abs/pii/S0091679X20301242?utm_source=chatgpt.com)
* **X10** — Truckenbrodt *et al.*, *EMBO Rep.* (2018) and *Nat. Protoc.* (2019). \~10× gels, \~25–30 nm effective resolution. [EMBO Press](https://www.embopress.org/doi/10.15252/embr.201845836?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/s41596-018-0117-3?utm_source=chatgpt.com), [publications.goettingen-research-online.de](https://publications.goettingen-research-online.de/bitstream/2/59786/2/s41596-018-0117-3.pdf?utm_source=chatgpt.com)
* **ExPath / rExPath (clinical pathology)** — Zhao *et al.*, *Nat. Biotechnol.* (2017) + protocol resources. FFPE-friendly workflows. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5548617/?utm_source=chatgpt.com), [Springer Nature Experiments](https://experiments.springernature.com/articles/10.1038/s41596-020-0300-1?utm_source=chatgpt.com), [JoVE](https://www.jove.com/t/60195/nanoscopic-imaging-human-tissue-sections-via-physical-isotropic?utm_source=chatgpt.com)
* **eMAP (epitope-preserving MAP)** — Park *et al.*, *Sci. Adv.* (2021). Physical hybridization to retain antigenicity. [Science](https://www.science.org/doi/10.1126/sciadv.abf6589?utm_source=chatgpt.com)
* **LR-ExM (label-retention)** — Shi *et al.*, *J. Cell Biol.* (2021) + updated protocol (2024). Trifunctional anchors reduce signal loss; SNAP/CLIP-tag friendly. [RUPress](https://rupress.org/jcb/article/220/9/e202105067/212454/Label-retention-expansion-microscopyLabel?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/34228783/?utm_source=chatgpt.com), [Current Protocols](https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.973?utm_source=chatgpt.com)
* **Pan-ExM** — Bewersdorf lab and applications (e.g., NPC plasticity in *J. Cell Biol.*, 2024). Bulk proteome staining for ultrastructural contrast. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11429769/?utm_source=chatgpt.com), [RUPress](https://rupress.org/jcb/article/224/9/e202409120/278050/Visualizing-nuclear-pore-complex-plasticity-with?utm_source=chatgpt.com)
## Recent advances (2022-2025)
* **TREx (Ten-fold Robust ExM)** — Damstra *et al.*, *eLife* (2022) (+bio-protocol 2023). Simple, single-step \~10× with membrane/total-protein context. [eLife](https://elifesciences.org/articles/73775?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/35179128/?utm_source=chatgpt.com), [en.bio-protocol.org](https://en.bio-protocol.org/en/bpdetail?id=4698&type=0&utm_source=chatgpt.com)
* **Magnify** — Klimas *et al.*, *Nat. Biotechnol.* (2023). “Universal” anchoring (proteins, nucleic acids, lipids), up to \~11×, FFPE-compatible. [Nature](https://www.nature.com/articles/s41587-022-01546-1?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/36593399/?utm_source=chatgpt.com), [magnify.mcs.cmu.edu](https://magnify.mcs.cmu.edu/?utm_source=chatgpt.com)
* **ExR (Expansion Revealing)** — Sarkar *et al.*, *Nat. Methods* (2022). Protein decrowding to unmask nanostructures (\~20 nm). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9551354/?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/36038771/?utm_source=chatgpt.com)
* **dExPath (decrowding ExPath)** — Valdes *et al.*, *Sci. Transl. Med.* (2024) (earlier preprints 2021–2022). Improved epitope access in human brain pathology. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10911838/?utm_source=chatgpt.com), [BioRxiv](https://www.biorxiv.org/content/10.1101/2021.12.05.471271v1?utm_source=chatgpt.com)
* **ONE microscopy** — Shaib *et al.*, *Nat. Biotechnol.* (2024). “One-step nanoscale expansion,” demonstrating \~1-nm-scale protein shape readouts and clinical CSF aggregates. [Nature](https://www.nature.com/articles/s41587-024-02431-9?utm_source=chatgpt.com)
* **Single-shot 20× ExM** — Wang *et al.*, *Nat. Methods* (2024). 20-fold expansion in a single round. [Nature](https://www.nature.com/articles/s41592-024-02454-9?utm_source=chatgpt.com) Yields 20 nanometer or better resolution on a conventional microscope using a single expansion step.
* **HiExM (high-throughput ExM)** — Day *et al.*, *eLife* (2024). 96-well plate pipeline for screening-style pipelines. [eLife](https://elifesciences.org/articles/96025?utm_source=chatgpt.com)
* **BOOST (hour-scale 10×)** — Guo *et al.*, *Nat. Commun.* (2025). Fast, single-step \~10× for cells, tissues, and FFPE. [Nature](https://www.nature.com/articles/s41467-025-57350-3?utm_source=chatgpt.com)
* **GelMap** — Damstra *et al.*, *Nat. Methods* (2023). Built-in fluorescent grids for expansion factor mapping, deformation correction, and correlative workflows. [Nature](https://www.nature.com/articles/s41592-023-02001-y?utm_source=chatgpt.com), [Springer Nature Experiments](https://experiments.springernature.com/articles/10.1038/s41592-023-02001-y?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/37723243/?utm_source=chatgpt.com)
## Good reviews & overviews
* **Chemical deep-dive review** — Wen *et al.*, *Chem. Rev.* (2023). Chemistries for grafting, gels, labeling strategies, fixation effects; broad survey. [American Chemical Society Publications](https://pubs.acs.org/doi/10.1021/acs.chemrev.2c00711?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/36881995/?utm_source=chatgpt.com)
* **Practical cell-biology guide** — Hümpfer & Sauer, *J. Cell Sci.* (2024). Accessible, method-choosing guidance and pitfalls. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11058692/?utm_source=chatgpt.com), [The Company of Biologists Journals](https://journals.biologists.com/jcs/article/137/7/jcs260765/346856/Expanding-boundaries-a-cell-biologist-s-guide-to?utm_source=chatgpt.com)
* **10-year technology feature** — Nature (2025). Where ExM stands, adoption, impact. [Nature](https://www.nature.com/articles/d41586-025-00059-6?utm_source=chatgpt.com)
* **Applied Physics Reviews perspective** — *APR* (2025). Broad overview of ExM for biological systems. [AIP Publishing](https://pubs.aip.org/aip/apr/article/12/2/021311/3345282/Nanoscale-imaging-of-biological-systems-via?utm_source=chatgpt.com)
* **Neuroscience-focused review** — Behzadi *et al.*, *Front. Neural Circuits* (2025). ExM in genetic animal models/circuits. [PubMed](https://pubmed.ncbi.nlm.nih.gov/39712646/?utm_source=chatgpt.com)
## Quick “when to use what”
* Need **max resolution** with simple optics → **iExM / single-shot 20× / ONE** (depending on sample and labeling). [Nature](https://www.nature.com/articles/nmeth.4261?utm_source=chatgpt.com)
* Need **ultrastructure context** → **U-ExM** or **pan-ExM**. [PubMed](https://pubmed.ncbi.nlm.nih.gov/30559430/?utm_source=chatgpt.com), [RUPress](https://rupress.org/jcb/article/224/9/e202409120/278050/Visualizing-nuclear-pore-complex-plasticity-with?utm_source=chatgpt.com)
* Need **FFPE/clinical** → **ExPath/dExPath** or **Magnify**. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5548617/?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/36593399/?utm_source=chatgpt.com)
* Need **fast or single-step large factors** → **TREx**, **BOOST**, **12×/20× single-shot**. [eLife](https://elifesciences.org/articles/73775?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/s41467-025-57350-3?utm_source=chatgpt.com)
* Need **quality control / correlative reg** → **GelMap**. [Nature](https://www.nature.com/articles/s41592-023-02001-y?utm_source=chatgpt.com)
## Advanced expansion microscopy methods
* **Magnify** (Klimas et al., 2023) — robust \~up to 11× expansion; retains proteins, nucleic acids, lipids. [Nature](https://www.nature.com/articles/s41587-022-01546-1?utm_source=chatgpt.com)
* **ONE microscopy** (Shaib et al., 2024) — one-step nanoscale ExM, down to \~1 nm effective resolution on processed material. [Nature](https://www.nature.com/articles/s41587-024-02431-9?utm_source=chatgpt.com)
* **TREx** (Damstra et al., 2022) — ten-fold robust expansion in a single round. [eLife](https://elifesciences.org/articles/73775?utm_source=chatgpt.com)
* **iU-ExM** (Louvel et al., 2023) — iterative ultrastructure ExM achieving SMLM-level resolution. [Nature](https://www.nature.com/articles/s41467-023-43582-8?utm_source=chatgpt.com)
* **HiExM** (preprint 2024) — high-throughput ExM in 96-well plates for cultured cells (fixed). [eLife](https://elifesciences.org/reviewed-preprints/96025v3?utm_source=chatgpt.com)
## Whole organism ExM
These are methods for expansion microscopy of fixed whole organisms.
* **Zebrafish ExM** (Freifeld et al., 2017) — protocols for intact fish tissues. [PNAS](https://www.pnas.org/doi/10.1073/pnas.1706281114?utm_source=chatgpt.com)
* **C. elegans ExM (ExCel)** (Yu et al., 2020) — expands fixed, intact worms despite the cuticle barrier. [eLife](https://elifesciences.org/articles/46249?utm_source=chatgpt.com)
## In vivo?
### No expansion microscopy for in vivo living systems
ExM’s core steps conflict with life: covalent anchoring and in-situ polymerization, extensive protease/denaturation to homogenize the matrix, membrane fragmentation, and large osmotic swelling—all fundamentally incompatible with intact, living cells/tissues. [BioMed Central](https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-017-0393-3?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11058692/?utm_source=chatgpt.com)
* Hümpfer & Sauer (2024), *A cell biologist’s guide to expansion microscopy* — clear statement that ExM involves fixation and is incompatible with live cells. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11058692/?utm_source=chatgpt.com)
* Gao & Boyden (2017), *Q\&A: Expansion microscopy* — direct answer “Does ExM work on live specimens? **No**,” with rationale (membranes fragment, proteins get diluted/anchored, etc.). [BioMed Central](https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-017-0393-3?utm_source=chatgpt.com)
* Behzadi et al. (2024/2025), *Expansion microscopy reveals neural circuit organization…* (review) — applications across animal models, all on fixed samples. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11660448/?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/39712646/?utm_source=chatgpt.com)
### Closest things to “live/in vivo” but not ExM
* **Correlative live-cell + ExM workflows** — image live first, then fix & expand; GelMap provides intrinsic calibration to find the same cells post-expansion. Useful if you want dynamics *and* nanoscale structure on the same cells, but the expansion step is after fixation. [Nature](https://www.nature.com/articles/s41592-023-02001-y?utm_source=chatgpt.com)
* **Transparent/electrophysiology hydrogels in vivo** — soft, transparent hydrogel interfaces enabling two-photon imaging and ECoG recordings in live mice (a materials route to better optical/electrode access, not cellular enlargement). [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S1742706122005256?utm_source=chatgpt.com)
* **Long-term, in vivo-stable hydrogels** — implanted gels tracked over 400 days in mice (biocompatibility context for in-body polymers). Again, not ExM. [Nature](https://www.nature.com/articles/s43246-025-00830-2?utm_source=chatgpt.com)
* **Live-cell “spacing” without killing** — *Flat Cell Imaging* physically flattens living cells to increase separations for high-res imaging. (In vitro; not in vivo and not ExM.) [arXiv](https://arxiv.org/abs/2411.12656?utm_source=chatgpt.com)
### For validation of electrode placement
Given the above, current practice is to (a) use ex vivo ExM (expansion microscopy) to design/validate nanoscale electrode geometries against true cellular ultrastructure, then (b) switch to in vivo-compatible soft/transparent interfaces (e.g., hydrogels) for live recordings/manipulations. The Wei et al. hydrogel paper is a good starting point for that materials track. [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S1742706122005256?utm_source=chatgpt.com)
## More references
* Klimas et al., 2023 — *Magnify*. [Nature](https://www.nature.com/articles/s41587-022-01546-1?utm_source=chatgpt.com)
* Shaib et al., 2024 — *ONE microscopy*. [Nature](https://www.nature.com/articles/s41587-024-02431-9?utm_source=chatgpt.com)
* Damstra et al., 2022 — *TREx*. [eLife](https://elifesciences.org/articles/73775?utm_source=chatgpt.com)
* Damstra et al., 2023 — *GelMap* for correlative live-cell + ExM. [Nature](https://www.nature.com/articles/s41592-023-02001-y?utm_source=chatgpt.com)
* Wei et al., 2022 — Transparent electrophysiological hydrogel for in vivo imaging + ECoG. [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S1742706122005256?utm_source=chatgpt.com)
* Kolouchova et al., 2025 — Long-term tracking of implanted hydrogels in mice. [Nature](https://www.nature.com/articles/s43246-025-00830-2?utm_source=chatgpt.com)
[US Patent - US20160304952A1 - In situ nucleic acid sequencing of expanded biological samples](https://patents.google.com/patent/US20160304952A1/en)
# See also
[[fisseq]]
|
