Expansion Microscopy is Transforming Biological Imaging

First reported in 2015, expansion microscopy (ExM) enables researchers to image cellular features that could previously only be seen with techniques like super-resolution microscopy (SRM) or electron microscopy (EM). Since its development, various expansion microscopy protocols have been published to cover a growing number of applications. 

Traditional light microscopy is unable to resolve features that are closer than 200 nm due to Abbe’s diffraction limit, a fundamental principle in optics which is determined by the wavelength (λ) of light used and the numerical aperture (NA) of the imaging system. Although techniques such as super-resolution microscopy (SRM) and electron microscopy (EM) can bypass this limit, they require specialized instrumentation and considerable technical expertise, which makes them inaccessible to many laboratories. 

Expansion microscopy, developed in the Boyden Laboratory at Massachusetts Institute of Technology (MIT), represents an easier way to improve the optical resolution¹. Instead of relying on electron beams like EM, or manipulating the light’s interaction with the sample like SRM, expansion microscopy uses water-swellable polymer gels to physically expand fixed biological material and allow for visualization with conventional light microscopes and common fluorescent reagents. Importantly, expansion microscopy can be readily adopted by researchers familiar with standard IHC/ICC staining protocols.  

In their seminal publication, Boyden et al. reported a method based on five main steps:

1) fluorescent labeling of fixed and permeabilized cells/tissue sections

2) sample infusion with polymer components followed by gelation, 3) protease digestion to allow for uniform expansion

4) dialysis in water

5) imaging.

This approach resulted in a 4.5-fold linear expansion, equating to approximately 70 nm lateral resolution, with a direct comparison of pre-ExM superresolution images to post-ExM confocal images confirming that there was no significant sample distortion. 

The original method has since evolved to both increase the resolution and improve the signal strength, which can often be impaired by epitope masking during gelation or protein loss upon digestion. In addition, the custom-made fluorescent reagents originally used by Boyden et al. have been superseded by conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins². 

Newer versions of ExM include Iterative Expansion Microscopy (iExM), which achieves 25 nm resolution after two successive rounds of expansion, and X10 Expansion Microscopy, which provides similar resolution with a single expansion step^3,4. Another method, known as ultrastructure expansion microscopy (U-ExM), has been combined with super-resolution microscopy to reveal cellular details that could otherwise be observed only by electron microscopy⁵. 

Expansion microscopy protocols can be broadly divided into two main categories according to when the labeling step takes place. Pre-expansion labeling involves staining the biomolecules and then expanding the tissue, while in post-expansion labeling these two steps are switched. Post-expansion labeling is often preferred when studying analytes with low abundance since it effectively increases the fluorophore concentration at the target site to boost the signal intensity. Alternatively, signal amplification can be accomplished using methods such as Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), which employs DNA-barcoded antibodies and orthogonal DNA concatemers for rapid, highly multiplexed super-resolution tissue imaging⁶. 

Although expansion microscopy is compatible with a broad array of commercial fluorescently-labeled antibodies and genetically encoded fluorophores, there are several notable exceptions. These include Cy5 and Alexa Fluor® 647, which are degraded during the polymerization step, and the bacteriophytochrome infrared protein iRFP, which is degraded by proteinase K digestion⁷. Researchers should always perform rigorous protocol optimization for their specific model system using appropriate positive and negative controls, and referring to the literature for guidance. 

Expansion microscopy has seen the greatest uptake for neuroscience research, where it allows for imaging intricate neuronal structures such as presynaptic terminals. Moreover, because expansion microscopy renders samples transparent, it is useful for visualizing whole intact organs, including the murine brain⁸. Other applications of expansion microscopy include its use to study centrosome biogenesis and maintenance, interrogate the biology of miniscule parasites, and visualize viral infection of human cells. 

LubioScience represents some of the most trusted brands in research and works closely with our partners to offer a broad range of high-quality products for established and emerging techniques. Our portfolio includes HAK-actin™ from Spirochrome, which ensures reliable staining of actin with U-ExM based protocols, and an extensive selection of IHC/ICC reagents from VectorLabs. 

1. Chen F, Tillberg PW, Boyden ES. Optical imaging. Expansion microscopy. Science. 2015;347(6221):543-548. doi:10.1126/science.1260088 

1. Tillberg PW, Chen F, Piatkevich KD, et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol. 2016;34(9):987-992. doi:10.1038/nbt.3625 

1. Chang JB, Chen F, Yoon YG, et al. Iterative expansion microscopy. Nat Methods. 2017;14(6):593-599. doi:10.1038/nmeth.4261 

1. Truckenbrodt S, Maidorn M, Crzan D, et al. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep. 2018;19(9):e45836. doi:10.15252/embr.201845836 

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

1. Saka SK, Wang Y, Kishi JY, et al. Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues. Nat Biotechnol. 2019;37(9):1080-1090. doi:10.1038/s41587-019-0207-y 

1. Gaudreau-Lapierre A, Mulatz K, Béïque JC, Trinkle-Mulcahy L. Expansion microscopy-based imaging of nuclear structures in cultured cells. STAR Protoc. 2021;2(3):100630. Published 2021 Jun 26. doi:10.1016/j.xpro.2021.100630 

1. Ku T, Swaney J, Park JY, et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat Biotechnol. 2016;34(9):973-981. doi:10.1038/nbt.3641