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
  • Gao & Boyden, 2017 — Q&A: Expansion microscopy. BioMed Central
  • Behzadi et al., 2024/2025 — ExM in genetic animal models (review). PMC, arXiv
  • Louvel et al., 2023 — iU-ExM. Nature
  • Freifeld et al., 2017 — Zebrafish ExM. PNAS

  • Yu et al., 2020 — C. elegans ExM (ExCel). eLife

  • ExSeq (Science, 2021) — expansion microscopy + in situ sequencing gives nanoscale-resolved RNA maps. Science, PMC

One-step nanoscale expansion microscopy reveals individual protein shapes 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, PMC
  • proExM (protein-retention) — Tillberg et al., Nat. Biotechnol. (2016). Anchors proteins directly to the gel; compatible with antibodies/FPs. PubMed
  • MAP (Magnified Analysis of the Proteome) — Ku et al., Nat. Biotechnol. (2016). 4× expansion of intact tissues with proteome preservation. PubMed, PMC
  • iExM (iterative ExM) — Chang et al., Nat. Methods (2017). Serial expansions toward \~20×. Nature, PubMed

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, ScienceDirect
  • X10 — Truckenbrodt et al., EMBO Rep. (2018) and Nat. Protoc. (2019). \~10× gels, \~25–30 nm effective resolution. EMBO Press, Nature, publications.goettingen-research-online.de
  • ExPath / rExPath (clinical pathology) — Zhao et al., Nat. Biotechnol. (2017) + protocol resources. FFPE-friendly workflows. PMC, Springer Nature Experiments, JoVE
  • eMAP (epitope-preserving MAP) — Park et al., Sci. Adv. (2021). Physical hybridization to retain antigenicity. Science
  • LR-ExM (label-retention) — Shi et al., J. Cell Biol. (2021) + updated protocol (2024). Trifunctional anchors reduce signal loss; SNAP/CLIP-tag friendly. RUPress, PubMed, Current Protocols
  • Pan-ExM — Bewersdorf lab and applications (e.g., NPC plasticity in J. Cell Biol., 2024). Bulk proteome staining for ultrastructural contrast. PMC, RUPress

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, PubMed, en.bio-protocol.org
  • Magnify — Klimas et al., Nat. Biotechnol. (2023). “Universal” anchoring (proteins, nucleic acids, lipids), up to \~11×, FFPE-compatible. Nature, PubMed, magnify.mcs.cmu.edu
  • ExR (Expansion Revealing) — Sarkar et al., Nat. Methods (2022). Protein decrowding to unmask nanostructures (\~20 nm). PMC, PubMed
  • dExPath (decrowding ExPath) — Valdes et al., Sci. Transl. Med. (2024) (earlier preprints 2021–2022). Improved epitope access in human brain pathology. PMC, BioRxiv
  • ONE microscopy — Shaib et al., Nat. Biotechnol. (2024). “One-step nanoscale expansion,” demonstrating \~1-nm-scale protein shape readouts and clinical CSF aggregates. Nature
  • Single-shot 20× ExM — Wang et al., Nat. Methods (2024). 20-fold expansion in a single round. Nature 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
  • BOOST (hour-scale 10×) — Guo et al., Nat. Commun. (2025). Fast, single-step \~10× for cells, tissues, and FFPE. Nature
  • GelMap — Damstra et al., Nat. Methods (2023). Built-in fluorescent grids for expansion factor mapping, deformation correction, and correlative workflows. Nature, Springer Nature Experiments, PubMed

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, PubMed
  • Practical cell-biology guide — Hümpfer & Sauer, J. Cell Sci. (2024). Accessible, method-choosing guidance and pitfalls. PMC, The Company of Biologists Journals
  • 10-year technology feature — Nature (2025). Where ExM stands, adoption, impact. Nature
  • Applied Physics Reviews perspective — APR (2025). Broad overview of ExM for biological systems. AIP Publishing
  • Neuroscience-focused review — Behzadi et al., Front. Neural Circuits (2025). ExM in genetic animal models/circuits. PubMed

Quick “when to use what”

  • Need max resolution with simple optics → iExM / single-shot 20× / ONE (depending on sample and labeling). Nature
  • Need ultrastructure context → U-ExM or pan-ExM. PubMed, RUPress
  • Need FFPE/clinical → ExPath/dExPath or Magnify. PMC, PubMed
  • Need fast or single-step large factors → TREx, BOOST, 12×/20× single-shot. eLife, Nature
  • Need quality control / correlative reg → GelMap. Nature

Advanced expansion microscopy methods

  • Magnify (Klimas et al., 2023) — robust \~up to 11× expansion; retains proteins, nucleic acids, lipids. Nature
  • ONE microscopy (Shaib et al., 2024) — one-step nanoscale ExM, down to \~1 nm effective resolution on processed material. Nature
  • TREx (Damstra et al., 2022) — ten-fold robust expansion in a single round. eLife
  • iU-ExM (Louvel et al., 2023) — iterative ultrastructure ExM achieving SMLM-level resolution. Nature
  • HiExM (preprint 2024) — high-throughput ExM in 96-well plates for cultured cells (fixed). eLife

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
  • C. elegans ExM (ExCel) (Yu et al., 2020) — expands fixed, intact worms despite the cuticle barrier. eLife

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, PMC

  • Hümpfer & Sauer (2024), A cell biologist’s guide to expansion microscopy — clear statement that ExM involves fixation and is incompatible with live cells. PMC
  • 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
  • Behzadi et al. (2024/2025), Expansion microscopy reveals neural circuit organization… (review) — applications across animal models, all on fixed samples. PMC, PubMed

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
  • 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
  • Long-term, in vivo-stable hydrogels — implanted gels tracked over 400 days in mice (biocompatibility context for in-body polymers). Again, not ExM. Nature
  • 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

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

More references

  • Klimas et al., 2023 — Magnify. Nature
  • Shaib et al., 2024 — ONE microscopy. Nature
  • Damstra et al., 2022 — TREx. eLife
  • Damstra et al., 2023 — GelMap for correlative live-cell + ExM. Nature
  • Wei et al., 2022 — Transparent electrophysiological hydrogel for in vivo imaging + ECoG. ScienceDirect
  • Kolouchova et al., 2025 — Long-term tracking of implanted hydrogels in mice. Nature

US Patent - US20160304952A1 - In situ nucleic acid sequencing of expanded biological samples

See also

fisseq