Getting to Know Expansion Microscopy

Expansion Microscopy (ExM) promises an easier, more accessible way to image biological features previously only visible via techniques like super-resolution microscopy or electron microscopy. Since its introduction in 2015 by the Boyden Lab, ExM has been steadily growing in popularity with an abundance of protocols and examples of applications from across disciplines now available. In this post, we’ll review the inspiration behind ExM, how it works, and some of the limitations to keep in mind whether you are planning your own ExM experiment or evaluating ExM images in a new publication.        

Why Expansion Microscopy? 

Traditional light microscopes are limited in the level of resolution that they can achieve. This means that regardless of how powerful of an objective you have, you can’t resolve two points within ~250 nm of each other. For reference, individual presynaptic terminals, which are jam packed with everything needed for synaptic transmission, are right around 250 nm, making it difficult to study the inner workings of the synapse by conventional light or fluorescence microscopy (Dani, et al., 2010).

How do you overcome this resolution limit? Electron Microscopy (EM) is one option, but the equipment needed for EM is not something you find in the average cell and molecular biology lab, and EM is limited in the number of specific targets you can label in a sample. Super-resolution microscopy (SRM), a family of imaging techniques developed to break through the resolution barrier while still utilizing fluorescence microscopy, is another option. However, most SRM techniques also require specialized instruments and technical expertise, limiting their accessibility (Prakash K, et al. 2022).

So what’s a researcher to do if they want to understand the ultrastructural relationships that influence everything from cell division to memory, but don’t have access to the specialized equipment needed for EM or SRM? Enter Expansion Microscopy.

What is Expansion Microscopy? 

ExM provides a straightforward answer to the space problem. Instead of imaging smaller, what if we just made the sample bigger? In ExM, and its various derivatives, biological samples are enmeshed in a gel of a highly absorbent material that is able to expand evenly in all directions to several times its original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This expansion physically separates molecules while maintaining their relative positions, allowing you to resolve individual points with a conventional, diffraction-limited light microscope. 

Along with using more accessible imaging equipment, ExM protocols utilize relatively inexpensive reagents and are readily adoptable by labs already familiar with IHC/ICC protocols. A typical ExM protocol (Fig. 1) follows this structure:

• Step 1: fix (and maybe label) your sample (i.e. cultured cells or tissues). 
• Step 2: attach anchors to your sample that will link biomolecules to the gel and add gel components to polymerize.
• Step 3: digest the sample to allow for even expansion. 
• In some cases, labels are added after this stage. 
• Step 4: Expand! (Typically by just adding water.) 
• Step 5: Image
  

Figure 1: The ExM process allows you to physically magnify a sample so you don’t have to optically magnify quite so much. Created with biorender.com

Steps 2-4 are not found in standard IHC/ICC protocols and you might expect them to dramatically limit the types of labels you could use. But many ExM protocols are compatible with conventional antibodies, chemical dyes, and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al. 2016). That being said, the anchoring, digestion, and expansion steps can impact the integrity of different targets and labels. You may want to check out a few different protocols to look for strategies that may be better for your specific targets.

What are some of the limitations? 

A “super-resolution” technique that uses common reagents, straightforward protocols, and standard microscopes sounds great, right? But there is always a catch - some limitation of the technique that users should be aware of - and ExM is no exception. First, expansion requires fixed samples. This limitation may be the both most obvious and least flexible one. Tissue expansion is not compatible with in vivo applications and there’s just not much to be done about that. 

Second, expansion introduces distortions. The goal in expansion is for your sample to expand evenly in all directions, aka isotropically. But achieving isotropic expansion depends on how well the gel components penetrate the sample, how well the gel polymerizes, and the level of digestion. All of these factors in turn depend upon the specific gel recipe used, ambient temperatures, incubation times, and the physical properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain slices, which are easier to expand than, say, a C. elegans (Damstra, et al. 2022; Yu, et al. 2020). With any variation, your samples can tear or expand unevenly, introducing distortions in your final images. It is possible to keep distortions to a minimum with protocol optimization and careful execution, but they cannot be entirely eliminated. It is recommended that you image samples before and after expansion and measure the degree of agreement between them to ensure the expanded images are still a good representation of reality.

Third, expansion results in dimmer samples. Reduced signal can be caused by loss of protein during digestion, or masking of epitopes during sample anchoring and gel polymerization - issues that can be minimized through tweaks to these steps. But even under ideal polymerization and digestion conditions, loss of signal often occurs, due to a reduction in target concentration during expansion. One group has found that using Fab fragment secondaries, rather than traditional full IgG secondaries, can improve signal strength (Sherry and Stiles, 2022).

Fourth, signal continuity can appear reduced in ExM, which could impact how you interpret a target’s structure. For example, cytoskeletal structures that form continuous lines in pre-expanded samples can sometimes take on a fragmented appearance post-expansion, but that doesn’t mean we should start re-conceptualizing actin filaments or microtubules. This issue is shared by other SRM techniques and can be alleviated by maximizing label density. In some cases, nanobodies have been found to help improve label density (Mikhaylova, et al. 2015). 

Despite these limitations, ExM is a valuable tool, especially when paired with complementary experimental approaches. Since it arrived on the scene, ExM has helped researchers gain a deeper understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux, et al. 2021; Gambarotto, et al. 2019; M’Saad, et al. 2020). Plus, it’s been demonstrated to work in different model systems, paired with SRM techniques, and adapted for detecting nucleic acids as well as proteins (Klimas, et al. 2023; Wassie, et al. 2019). Perhaps it’s the tool you’ve been looking for too!    

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References and resources

References

Bertiaux E, Balestra AC, Bournonville L, et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid. PLoS Biology 19:e3001020. https://doi.org/10.1371/journal.pbio.3001020 

Chen F, Tillberg PW, Boyden ES (2015) Expansion microscopy. Science 347:543–548. https://doi.org/10.1126/science.1260088

Damstra HG, Mohar B, Eddison M, et al (2022) Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). Elife 11:e73775. https://doi.org/10.7554/elife.73775 

Dani A, Huang B, Bergan J, et al (2010) Superresolution Imaging of Chemical Synapses in the Brain. Neuron 68:843–856. https://doi.org/10.1016/j.neuron.2010.11.021 

Gambarotto D, Zwettler FU, Guennec ML, et al (2019) Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat Methods 16:71–74. https://doi.org/10.1038/s41592-018-0238-1 

Klimas A, Gallagher BR, Wijesekara P, et al (2023) Magnify is a universal molecular anchoring strategy for expansion microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1 

Mikhaylova M, Cloin BMC, Finan K, et al (2015) Resolving bundled microtubules using anti-tubulin nanobodies. Nat Commun 6:7933. https://doi.org/10.1038/ncomms8933 

M’Saad O, Bewersdorf J (2020) Light microscopy of proteins in their ultrastructural context. Nat Commun 11:3850. https://doi.org/10.1038/s41467-020-17523-8

Prakash K, Diederich B, Heintzmann R, Schermelleh L (2022) Super-resolution microscopy: a brief history and new avenues. Philosophical Transactions Royal Soc 380:20210110. https://doi.org/10.1098/rsta.2021.0110 

Sherry DM, Stiles MA (2022) Improved fluorescent signal in expansion microscopy using fluorescent Fab fragment secondary antibodies. Methodsx 9:101796. https://doi.org/10.1016/j.mex.2022.101796 

Tillberg PW, Chen F, Piatkevich KD, et al (2016) Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34:987–992. https://doi.org/10.1038/nbt.3625 

Truckenbrodt S, Sommer C, Rizzoli SO, Danzl JG (2019) A practical guide to optimization in X10 expansion microscopy. Nat Protoc 14:832–863. https://doi.org/10.1038/s41596-018-0117-3

Wassie AT, Zhao Y, Boyden ES (2019) Expansion microscopy: principles and uses in biological research. Nat Methods 16:33–41. https://doi.org/10.1038/s41592-018-0219-4 

Yu C-C (Jay), Barry NC, Wassie AT, et al (2020) Expansion microscopy of C. elegans. Elife 9:e46249. https://doi.org/10.7554/elife.46249 

Additional resources on the Addgene blog

• Antibodies 101: Introduction to Immunofluorescence 
• Antibodies 101: Multiplex Immunofluorescence
• Antibodies 101: Affinity Reagents