Advances in Expansion Microscopy

Expansion microscopy was introduced in 2015 by Boyden and his team, revolutionizing the way we see biological samples under a microscope¹. Taking advantage of the power of physical enlargement, ExM pushes spatial resolution beyond the diffraction limit, allowing us to observe intricate details at previously unimaginable levels. The underlying principle of ExM lies in a clever combination of chemistry and physics. First, fluorescently labeled molecules are crosslinked to a hydrogel, which serves as a scaffold for the subsequent process. Then, the hydrogel is expanded isotropically with water, causing the sample to enlarge in all three dimensions. This isotropic expansion effectively separates the signals and enables higher-resolution imaging. Some of the most common ExM workflows are summarized in Figure 1 (taken from Zhuang Y. and Shi X. 2023²).

This method does not rely on optical or algorithmic improvements and can increase resolution by up to 4.5 times. By embedding fixed biological specimens in a hydrogel matrix and expanding them, structures as small as 70 nm can be resolved using conventional fluorescence microscopy. The expanded samples are optically clear, enabling imaging of thicker specimens that were previously difficult to visualize. The advantages of ExM are significant and were nicely described in Shoh Asano and Ruixuan Gao`s FocalPlane post from 2020 but a lot has happened since then!

Alternative labeling, gelation, and fixation protocols

An important milestone that facilitated the adoption of ExM in diverse biological fields was the adaptation of immunofluorescence labeling using conventional antibodies³. Nevertheless, its incompatibility with live sample imaging and the reduced signal due to the dispersed fluorophores in the larger sample size is a limitation. To circumvent this problem, many alternatives have arisen in the last few years. For example, label-retention expansion microscopy (LR-ExM) prevents signal loss and greatly enhances labeling efficiency using trifunctional anchors. This technique can be applied to immunofluorescence labeling and also to tagged proteins⁴. Another alternative is to use fluorescently labeled Fab fragment secondary antibodies to enhance the desired signal⁵. The use of stabilizer-containing organic fluorophores to mitigate the degradation of dyes due to free radicals has also been explored⁶. Another approach reported was repeatedly labeling target proteins with a pair of secondary antibodies that specifically bind to each other thus enhancing immunofluorescence signal⁷. As mentioned, fluorescent proteins can be used for ExM but they are often destroyed during sample preparation. To solve this problem, a method based on a DNA oligonucleotide linked with a fluorescent dye, an acrylamide group (linker), and benzoyl guanine has been reported⁸. Additionally, new chemically stable Red Fluorescent Proteins have been developed and applied in combination with ExM improving the labeling efficiency⁹.

Finally, it is very exciting that labeling other biomolecules for ExM is possible. Using click-labeling (Click-ExM) with biotin for example, and then staining with streptavidin conjugated to a fluorophore it is possible to track lipids, glycans, proteins, DNA, RNA, and small molecules¹⁰. Lipid expansion microscopy (LExM) has been developed to label phospholipids in cells and organelles membranes¹¹. Other interesting applications that label chromatin and epigenetic marks have been described, for example, ChromExM allows the visualization of chromatin, transcription, and transcription factors in vivo to study nuclear organization at nanoscale¹². It is also possible to generate spatial information between epigenetic readers and histone modifications (ExEpi)¹³.

One of the most remarkable features of ExM is its modular nature, offering flexibility to adapt to various biological samples. In the last couple of years, many alternative gelation protocols have been developed, for example, using vacuum stable gels that allow highly multiplexed staining and are also suitable for coupling with mass-spectrometry assays in tissues¹⁴. Another interesting alternative is photopolymerized hydrogels (PhotoExM), which allow for a faster processing time and maintain the integrity of the cells even in 3D cultures¹⁵. Also, the use of tetra-gel, a hydrogel that does not depend on free-radical chain-growth polymerization like commonly used polyacrylamide, has been proven useful to improve super-resolution imaging. Tetra-gel diminishes the labeling-placement distortions sometimes observed with polyacrylamide gels¹⁶. Finally, a cutting-edge technique called thermo-responsive reversible ExM (T-RevExM) allows the expansion factor to be thermally adjusted in a reversible manner according to the user requirements¹⁷.

Another step in the ExM protocol that can be tuned is the fixation approach which in some cases improves the ultrastructural preservation of the sample. For example, the use of cryofixation instead of chemical fixation has allowed the visualization of native cellular components and enhances the availability of epitopes, the downside is that it requires specific equipment¹⁸.

Making your samples even bigger and improving the resolution

Recently higher expansion factors have been reached, like Ten-fold Robust Expansion Microscopy (TREx) which allows 10-fold expansion with no specialized equipment of procedures¹⁹ or Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins, and lipids without the need for a separate anchoring step obtaining an 11-fold expansion²⁰.

Other approaches have relied on combining ExM with different microscopy techniques like light sheet microscopy (ExLLSM) to reconstruct complex 3D structures reducing imaging time, for example in neural circuits²¹. Also, an innovative technique called “Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and Stimulated Raman Scattering Microscopy” (MAGNIFIERS) combines stimulated Raman scattering microscopy with ExM, enabling label-free nanoscale imaging of biological targets like proteins, lipids, and DNA²².

In the pursuit of even greater resolution, researchers have combined ExM with super-resolution microscopy techniques. So far, ExM has been used in combination with structured illumination microscopy (ExSIM)²³, with stimulated emission depletion microscopy (ExSTED)²⁴ and single-molecule localization microscopy (Ex-SMLM and Ex-dSTORM)^25,26. This powerful synergy has pushed resolutions below 20 nm, revealing intricate details at the nanoscale level. More recent applications include the combination of 10-fold expansion microscopy with super-resolution radial fluctuations (SRRF), this new technique is called one-nanometer expansion (ONE) microscopy and allows the visualization of biomolecules at resolutions approaching 1 nm²⁷. ExSRRF has also been applied to pathology specimens²⁸. Furthermore, Klimas and colleagues have combined Magnify with super-resolution optical fluctuation imaging (SOFI) (preLights post from Nadja Hümpfer) and were able to clearly resolve fine details of cilia and basal bodies in human stem-cell-derived lung organoids, observing details previously visualized only with electron microscopy²⁰.

Imaging whole organisms and organs

Expansion microscopy is set to play a crucial role in visualizing and tracing large cellular morphologies and molecular components throughout entire organisms or organs. Recent developments in this direction include the adaptation of ExM to use in 3D organoid models that are difficult to image because they are hard to penetrate with conventional microscopes. Using phototransfer by allyl sulfide exchange-expansion microscopy (PhASE-ExM), Blatchley and colleagues were able to obtain clear samples and perform super-resolution imaging of organoids and their extracellular matrix in 3D²⁹.

Adaptations of ExM have allowed us to image whole organisms, including ExCel which allows the expansion of C. elegans achieving ~25 nm resolution³⁰, and TissUExM, a method to image whole vertebrate embryos that enables in situ mapping of protein complexes in different organelles³¹ (Figure 2). 

Recently ExM has expanded to plants and fungi, which have the additional complexity of the cell wall, a rigid structure resistant to conventional ExM denaturation and digestion processes^32,33. Microorganisms like some types of unicellular fungi and protozoa are sometimes difficult to image due to their small size and in the last couple of years, several ExM protocols have been reported to capture ultrastructural details in these organisms^34–38. Especially for protozoan parasites, like Trypanosoma cruzi the causative agent of Chagas disease, I have found ExM very useful to study the particularities of its cytoskeletal network and its single mitochondria³⁹. Very exciting progress has also been made in Plasmodium where the biogenesis and protein distribution of a variety of organelles and structures has been determined using ExM⁴⁰ (preLights post from Nadja Hümpfer).

Additionally, a recent preprint describes µMagnify, a method derived from ExM that allows imaging of pathogens and infected tissues using a universal biomolecular anchor. This method has a lot of potential for studying how microbes interact with their host systems and will be a powerful tool to develop new diagnosis and treatment strategies against infectious diseases⁴¹. It also could be very useful to study host-microbiota interactions.

What´s next?

For centuries, optical microscopy has been a cornerstone in histopathology and microbiology. ExM and its modifications have been tested extensively in different tissue and microbiological samples⁴². The idea that enlarging the sample could lead to enhanced diagnostic precision is very attractive, especially if we considered the prospect of ExM being adapted to a high-throughput format⁴³, accompanied by the advancement of image processing algorithms and computer power. Another interesting application that has implications for both biomedicine and material sciences is the adaptation of ExM to study cell-material interfaces, for example to study osseointegration of implants⁴⁴.

Finally, it is interesting to mention a new technique based on the ExM principle, called Unclearing Microscopy (preLights post from Robert Mahen), which allows expanding cells and tissues up to 8000-fold⁴⁵. This method labeles proteins using NHS-ester ligands linked to biotin in a non-selective manner. Then, they are subsequently exposed to streptavidin bound to horseradish peroxidase and a chromogenic substrate (like 3,3’-diaminobenzidine (DAB) or silver). The samples are then ready to be visualized with the naked eye and if imaged with transmitted light microscopes it is possible to observe details previously only accessible with super-resolution fluorescence or electron microscopy methods.

In conclusion, Expansion Microscopy has opened new frontiers in the world of microscopy, transcending the limits of conventional imaging techniques. Also, it democratizes access to super-resolution microscopy because even labs with standard equipment can set up an ExM workflow that adjusts to their biological sample and needs.

Acronyms for the different methods

References

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