Developments & applications
Expansion microscopy was developed in 2015 by Fei Chen, Paul W. Tillberg, and Ed Boyden (DOI: 10.1126/science.1260088). Since its invention, many variations have been made to improve the technique such as the Magnified Analysis of the Proteome (MAP) approach in 2016 which allows for volumetric expansion of the proteome (DOI: 10.1038/nbt.3641). In 2019, we developed ultrastructure expansion microscopy (U-ExM), an approach derived from the MAP protocol that allows optimal and isotropic expansion of cell architecture and macromolecules such as the centriole. Below you will find explanations and links to the different approaches we have developed-
ULTRASTRUCTURE EXPANSION MICROSCOPY (U-ExM)
Using the centriole as a molecular ruler, we optimized the MAP protocol to obtain an expansion factor of 4 that preserves at best the architecture of the cellular components. We tested this protocol on different organelles such as mitochondria, cytoskeleton or the phage T4.
CRYO-EXPANSION MICROSCOPY (Cryo-ExM)
U-ExM is a protocol developed for optimal cell expansion. However, this step requires that the cell be previously fixed chemically, which can affect the native architecture of the cell. By combining cryo-fixation, used in electron microscopy, and U-ExM, we have developed Cryo-ExM, a technique that maintains the organization of the cell before its expansion and thus allows the visualization of the cell in its quasi-native state.
ITERATIVE U-ExM (iU-ExM)
Expansion microscopy generally allows an expansion factor of 4. Despite numerous protocols allowing a larger expansion factor, it remains difficult to reach the same resolution than Single Molecule Light Microscopy. Here, we have developed an iterative U-ExM protocol allowing us to reach a resolution of the order of 10nm.
Link to the article : https://www.nature.com/articles/s41467-023-43582-8
U-ExM in tissue
U-ExM works very well for soft tissue, however, as the sample is thicker than cell monolayers, some steps require longer incubations. We have tested this approach for our research on retinitis pigmentosa.
U-ExM in Toxoplasma and Plasmodium
Apicomplexan parasites such as Toxoplasma gondii and Plasmodium possess highly specialized cytoskeletal structures that are difficult to resolve using conventional microscopy. In collaboration with the laboratories of Dominique Soldati-Favre and Mathieu Brochet, we pioneered the application of U-ExM to these parasites, revealing new details of their cytoskeletal organization, conoid architecture, and flagellar assembly.
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Membrane in Cryo-ExM
There are several techniques to see membranes in expansion microscopy but most of them require specific probes. Here, we have optimized the freeze substitution approach of our cryo-ExM protocol by adding some formaldehyde and paraformaldehyde in the process. Thanks to this approach, it is possible to visualize membranes simply by using the membrane marker BODIPY and mCLING.
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U-ExM in yeast
The U-ExM protocol does not require the use of proteinase K as in the original ExM protocol, making it very difficult to expand biological material with a cell wall that resists expansion. Here, by using an enzyme to digest the yeast cell wall, we have demonstrated that it is possible to use U-ExM and Cryo-ExM to visualize the architecture of S. pombe and S. cerevisiae.
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U-ExM in Chlamydomonas
Chlamydomonas is a green algae that our laboratory uses to study the centriole and the asembly of the flagella. To help the community to use U-ExM, we have written a step-by-step guide. In this protocol, we also show the effects of fixations, and above all we show that cryo-EXM coupled with BODIPY staining enables us to see membrane organelles and the thylakoids.
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U-ExM on C.elegans embryo
C. elegans is a widely used model organism to study cell biology and development. However, applying expansion microscopy to embryos has been challenging due to their protective eggshell. In this study, we show that combining freeze-cracking with U-ExM allows us to expand embryos fourfold while preserving their cellular architecture. Using nuclear pores as an example, we demonstrate that individual pores can be resolved, opening new possibilities for studying cellular structures during embryonic development.
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U-ExM in planktonic protists
Planktonic protists are incredibly diverse unicellular eukaryotes that play essential roles in marine ecosystems. Despite their ecological importance, their cellular organization remains poorly understood. In this study, in collaboration with the Dey and Dudin labs, we use U-ExM to explore the remarkable diversity of cytoskeletal architectures found in planktonic protists. By revealing intricate microtubule networks and previously unresolved cellular structures, we demonstrate how expansion microscopy can help us better understand the cellular complexity and evolution of these fascinating organisms.
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HAK-actin
Visualizing actin filaments using U-ExM has remained challenging due to the lack of reliable staining probes. In this study, we developed HAK-actin, a new probe specifically designed to label actin after expansion. We demonstrate that HAK-actin provides robust and uniform staining across a wide range of biological samples, including human cells, microbial eukaryotes, and mouse retinal tissue.
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U-ExM in FFPE clinical samples
Formalin-fixed paraffin-embedded (FFPE) tissues are widely used in clinical research, but their cellular ultrastructure remains difficult to resolve using conventional microscopy. In this study, in collaboration with the laboratories of Benita Wolf and Denis Migliorini, we demonstrate that expansion microscopy can be applied to FFPE human brain tumors to visualize tumor-infiltrating T cells and their cytotoxic granules.
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U-ExM in cryo-sections
Applying U-ExM to tissue sections opens new possibilities for studying cellular architecture directly in complex tissues. In this study, in collaboration with the laboratory of Yvan Arsenijevic, we used U-ExM on retinal cryo-sections to visualize the nanoscale organization of photoreceptor connecting cilia. This approach allowed us to assess structural defects caused by mutations in FAM161A and evaluate their restoration following gene therapy, highlighting the potential of U-ExM for investigating human diseases and therapeutic strategies.
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U-ExM in Human Motile Cilia
Human airway epithelial cells differentiated at the air–liquid interface (ALI) provide a powerful model to study motile cilia. Using U-ExM, we can map the nanoscale organization of proteins within the ciliary axoneme, transition zone, and basal bodies. Combined with cryo-electron tomography (cryo-ET), this approach reveals the molecular architecture of motile cilia and helps uncover structural defects associated with diseases such as primary ciliary dyskinesia (PCD).
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