What is expansion microscopy?

How does a microscopy technique like expansion microscopy (ExM) generate widespread adoption and lead to 700+ publications¹ after only being discovered 10 years prior? It solves a fundamental problem in the field of microscopy using a novel, yet accessible approach.

200 nanometers is the established resolution limit of conventional microscopy, which means that resolving the detailed features of two objects that are closer together than this distance is unattainable with standard methods.

Novel, yet expensive and/or highly specialized approaches such as electron microscopy and super resolution microscopy² have been developed to overcome this limit with great success. ExM also solves this resolution limitation, but takes a different approach, which is to make the object proportionally larger.

While proportionally enlarging something may seem simple, anyone who has tried to enlarge an image in PowerPoint by dragging one edge at a time quickly realizes how challenging this process is and how disproportionate their final image often becomes. Doing this in 3D with a cell or tissue sample appears even more daunting.

Nevertheless, the Boyden lab at MIT took on this challenge and developed a relatively simple and cost-effective approach in 2015, which he coined as ExM³.A fundamental principle of ExM is the use of a swellable polyelectrolyte hydrogel within a fixed specimen that, upon expansion, will physically pull the sample components apart, isotropically, which is to retain its spatial proportions in all directions with minimal distortions.

The result is enhanced resolution ranging from fourfold in the original manuscript to upwards of 20-fold linearly with iterative expansion microscopy methodologies⁴. The latest advancements have led to near single-molecule nanoscopic resolution, which we discuss below.

What is needed to perform expansion microscopy?

The initial expansion microscopy protocol utilized four core steps, including Anchoring, Gelation, homogenization, and Expansion (see Figure 1). Since then, the research community has incorporated changes such as different gel chemistries, fixation approaches, reaction conditions, and swelling procedures that have collectively increased sample expansion and pushed resolution to as low as one nanometer, but the core steps remain fairly constant.

Anchoring: The first step after fixing cells is to anchor the molecules of interest (e.g., protein, nucleic acids, lipids, etc.) to the hydrogel via covalent attachment.

Gelation: The next step, known as gelation, is to infuse the sample with the monomer solution of the hydrogel, which polymerizes to form a dense network that links to the anchored molecules of interest.

Homogenization: The sample is then homogenized/digested with enzymes or mechanical disruption to destabilize… 

Expansion: The fourth step involves submerging the sample in water, where it undergoes expansion, leading to swelling and transparency.

With the help of fluorescent probes, this reveals intricate molecular details when the samples are finally examined under the microscope.

This is a brief overview of the steps and more details are available here⁵. each of which must be carefully optimized to ensure the highest possible level of isotropy (sameness in all directions) and avoid introducing experimental artifacts.

While the core steps of ExM are relatively simple, inexpensive, and adoptable by other labs, there are clear drawbacks that should be highlighted. First and foremost, ExM only works with fixed samples and cannot be utilized in live cell applications. Furthermore, ExM is susceptible to distortions, requires optimization tailored to the experimental model, may result in dimmer samples, the process is time-consuming, and the signal continuity of larger structures may be disrupted. These drawbacks are discussed in more detail in our newsletter on ExM.

Therefore, it is essential to utilize proper controls when utilizing ExM. Comparing between images taken before and after expansion is necessary to control for isotropic expansion, and will help identify issues with distortion, signal continuity, and diminished reagent performance. Alternative control options are discussed here⁵.

How is the field of Expansion Microscopy Evolving?

One of the greatest features of ExM is that it can be adapted and optimized to overcome challenges and tailor the methodology to the investigator's model and experimental needs. This is evident in the growth of the field and the expansion of variations in ExM methodologies. One review highlights over 22 different methods that have been adapted for unique investigation, such as, ExM for lipid membranes (mExM with pGk5b and mCLING), RNA and DNA (Ex FISH), proteins (Pan-ExM), and other biomolecules⁵.

Furthermore, ExM has been combined with other innovative methods like super resolution microscopy^6-8, spatial transcriptomics⁹, and mass spectrometry imaging¹⁰. ExM is now being used for groundbreaking studies in Drosophila synaptonemal complexes¹¹, imaging whole mouse embryos¹² and zebrafish¹³, as well as, analyzing biopsies of cancer tissue⁹.

Intense effort is also being put toward enhancing expansion factors, developing improved hydrogels, and modifying homogenization approaches.

What are interesting applications and techniques for Expansion Microscopy?

As discussed above, there are numerous modified ExM methodologies that have been used for a plethora of applications to answer critical biological questions. Several have made a profound impact on the field, and we will summarize six groundbreaking or novel ExM applications, including: iExM, U-ExM, iU-ExM, Cryo-ExM, HiExM, and ONE.

Iterative Expansion Microscopy (iExM):

iExM is a modified version of ExM where the sample is exposed to a second swellable polymer mesh and expanded again⁴. iExM can expand biological samples to roughly 20 times their size in linear dimension, which results in 25 nm resolution imaging of tissues and cells with conventional microscopes.

The Boyden lab used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

Ultrastructure Expansion Microscopy (U-ExM):

One of the challenges in visualizing ultrastructures like organelles was that the original ExM methodology led to significant distortions. Thus, the Guichard and Hamel groups developed U-ExM by modifying the polymer chemistry, fixation, and labeling steps which better preserved the organelle structures¹⁴.

When combined with super-resolution approaches, it allowed for detailed visualization of centriolar chirality that was only possible with electron microscopy¹⁴. They have since gone on to perform a time-series reconstruction of the human centriole assembly using U-ExM¹⁵. This approach is compatible with other larger cell structures like mitochondria and tubulin.

Iterative Ultrastructural Expansion Microscopy (iU-ExM):

A challenge for ExM was whether it could achieve the same precision as nanoscopic techniques like single molecule localization microscopy. Louvel et al. show that it is possible, through their recently developed approach with they coined as iU-ExM. This combines iExM with U-ExM and produces SMLM-level resolution⁸. The group showed that iU-ExM is capable of visualizing molecular architecture of established structures like the eight-fold symmetry of nuclear pores.

With its wide-ranging applications, from isolated organelles to cells and tissue, iU-ExM opens new super-resolution avenues for scientists studying biological structures and functions.

Cryofixation Expansion Microscopy (Cryo-ExM):

Cryofixation preserves native cell ultrastructure without inducing many of the artifacts associated with chemical fixation; thus, making it an invaluable method for efficient preservation of native cell ultrastructure. In 2022, the Hamel and Guichard group developed Cryo-ExM which couples cryofixation with expansion microscopy¹⁶.

This methodology showed excellent preservation of the cytoskeletal landscape, integrity of organelles like the ER and mitochondria, as well as epitope accessibility¹⁶. Cryo-ExM shows that the cell ultrastructure is maintained through the cryofixation, freeze substitution, and ExM procedure, and circumvents the need to optimize chemical fixation processes and the potential associated artifacts.

High-throughput Expansion Microscopy (HiExM):

One of the limitations of ExM is the slide-based preparation and manual imaging, which caps the number of samples that can be processed and analyzed. The Boyer lab recently developed HiExM, which is a platform that enables ExM of cells cultured in a 96-well plate¹⁷. This approach enables fourfold expansion of wells within individual cells and can be combined with high-throughput confocal imaging platforms, enabling scalable super resolution imaging that may be valuable for drug studies, antibody development, and other screening-based applications.

One-step nanoscale expansion (ONE) microscopy:

As technologies are developed to push the boundary of what can be visualized by ExM approaches, none have enabled end users to visualize conformational changes in proteins with exquisite detail. The Rizzoli labs ONE microscopy method enables the visualization of changes in soluble proteins down to 1 nm resolution¹⁸. The approach combines X10 ExM with super-resolution radial fluctuations (SRRF) to circumvent issues associated with combining ExM with other super-resolution techniques.

Utilizing this approach, the group was able to report confirmation changes in single-molecule protein shapes; for example, they reported on changes in calmodulin upon Ca²⁺ binding. The authors state that ONE bridges the gap between structural biology techniques and light microscopy¹⁸.

Protocols

The entire process generally takes a day or two, with step-by-step protocols available at websites such as expansionmicroscopy.org

extensive reviews of the chemistry underlying ExM approaches have been published elsewhere (Truckenbrodt, 2023; Wen et al., 2023a) and detailed protocols are available (see Table S1).

References

1. Dolgin, E., 'Expansion microscopy' turns ten: how a tissue-swelling method brought super-resolution imaging to the masses. Nature, 2025. 637(8046): p. 752-754.

2. Prakash, K., et al., Resolution in super-resolution microscopy - facts, artifacts, technological advancements and biological applications. J Cell Sci, 2025. 138(10).

3. Chen, F., P.W. Tillberg, and E.S. Boyden, Optical imaging. Expansion microscopy. Science, 2015. 347(6221): p. 543-8.

4. Chang, J.B., et al., Iterative expansion microscopy. Nat Methods, 2017. 14(6): p. 593-599.

5. Humpfer, N., R. Thielhorn, and H. Ewers, Expanding boundaries - a cell biologist's guide to expansion microscopy. J Cell Sci, 2024. 137(7).

6. Halpern, A.R., et al., Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization. ACS Nano, 2017. 11(12): p. 12677-12686.

7. Zwettler, F.U., et al., Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Nat Commun, 2020. 11(1): p. 3388.

8. Louvel, V., et al., iU-ExM: nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy. Nat Commun, 2023. 14(1): p. 7893.

9. Alon, S., et al., Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems. Science, 2021. 371(6528).

10. Bai, Y., et al., Expanded vacuum-stable gels for multiplexed high-resolution spatial histopathology. Nat Commun, 2023. 14(1): p. 4013.

11. Cahoon, C.K., et al., Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex. Proc Natl Acad Sci U S A, 2017. 114(33): p. E6857-E6866.

12. Sim, J., et al., Nanoscale Resolution Imaging of Whole Mouse Embryos Using Expansion Microscopy. ACS Nano, 2025. 19(8): p. 7910-7927.

13. Freifeld, L., et al., Expansion microscopy of zebrafish for neuroscience and developmental biology studies. Proc Natl Acad Sci U S A, 2017. 114(50): p. E10799-E10808.

14. Gambarotto, D., et al., Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat Methods, 2019. 16(1): p. 71-74.

15. Laporte, M.H., et al., Time-series reconstruction of the molecular architecture of human centriole assembly. Cell, 2024. 187(9): p. 2158-2174 e19.

16. Laporte, M.H., et al., Visualizing the native cellular organization by coupling cryofixation with expansion microscopy (Cryo-ExM). Nat Methods, 2022. 19(2): p. 216-222.

17. Day, J.H., et al., High-throughput expansion microscopy enables scalable super-resolution imaging. Elife, 2024. 13.

18. Shaib, A.H., et al., One-step nanoscale expansion microscopy reveals individual protein shapes. Nat Biotechnol, 2025. 43(9): p. 1539-1547.