Expansion microscopy: A chemical approach for super-resolution microscopy

Zhuang, Yinyin; Shi, Xiaoyu

doi:10.1016/j.sbi.2023.102614

Cited by 18 publications

(6 citation statements)

References 65 publications

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“…Recent studies have explored the structural connectivity and functional interactions between dopaminergic neurons and specific central complex neuropils in Drosophila, including the FB and EB 24,25 . We employed the TH-Gal4>6xGFP Drosophila line [26][27][28] to assess the imaging quality of expanded fruit fly brain samples using Naked-ExM. In the TH-Gal4>6xGFP line, the dopaminergic neurons have been genetically engineered to express six GFP tags, enhancing their visibility through immunofluorescent labeling with GFP antibodies.…”

Section: Evaluating Naked-exm Imaging Quality Of Fruit Fly Brainsmentioning

confidence: 99%

Deployment of the natural magnifier CMTKG for naked expansion microscopy of biological tissues

Chen,

Tian,

Cheng

et al. 2024

Preprint

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Expansion Microscopy (ExM) enhances biological imaging to the nanoscale level by enlarging biological specimens, increasing the separation between biomolecules while maintaining their spatial arrangement. By integrating ExM with traditional microscopy, super-resolution volumetric images can be captured efficiently without compromising tissue integrity. Accordingly, fine molecular structures previously indiscernible by means of standard fluorescence microscopy can be visualized. However, one of the major challenges with ExM is that the samples are embedded in the polymerized gel, which limits the use of high numerical aperture objective lenses due to their short working distances. To address this issue, we have developed an approach using carboxymethyl tamarind kernel gum (CMTKG) cross-linked with sodium acrylate to create a superabsorbent hydrogel suitable for ExM. This technique facilitates removal of excess polymers from the expanded sample, resulting in sample exposure to the objective lens. Here, we demonstrate that it is possible to achieve approximately 7X expansion of Drosophila melanogaster brains, free from surrounding hydrogel, in a process we have named Naked-ExM. Followed by iterative expansion, the centimeter-sized fly brain is obtained and the ~15 um sized synapses are measured at 15X expansion.

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Section: Evaluating Naked-exm Imaging Quality Of Fruit Fly Brainsmentioning

confidence: 99%

Deployment of the natural magnifier CMTKG for naked expansion microscopy of biological tissues

Chen,

Tian,

Cheng

et al. 2024

Preprint

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“…Beyond cultured cells 18 , protocols have been validated for the gelation and expansion of organoids 19,20 , ex vivo tissue 21,22 , and several model organisms including C. elegans 23 . The resolution limits of ExM continue to improve through the identification of gel formulations and chemistries that support denser sample labeling and sturdier gels with higher swelling factors 18,24 .…”

Section: Introductionmentioning

confidence: 99%

Bio-orthogonal Glycan Imaging of Culture Cells and Whole AnimalC. eleganswith Expansion Microscopy

Kuo,

Colville,

Sorkin

et al. 2024

Preprint

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Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O-glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O-glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O-glycosylation in the first larval stage and identify O-glycan hotspots in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans, we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM.

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“…These methods require access to specific instrumentation, restricting superresolution imaging of membranes to laboratories with access to these specialized microscopes. Expansion microscopy (ExM) has emerged as an accessible technique that leverages sample expansion, rather than advanced microscope components, for superresolution imaging (Asano et al, 2018;Chen et al, 2015;Chozinski et al, 2016;Ku et al, 2016;Truckenbrodt, 2023;Wen et al, 2022;Wang et al, 2022;Zhuang & Shi, 2023). With this approach, samples are embedded into a swellable polymer network that facilitates physical expansion of ∼5×, effectively increasing the resolution limit to ∼50 nm.…”

Section: Introductionmentioning

confidence: 99%

Super‐Resolution Imaging of Clickable Lipids With Lipid Expansion Microscopy (LExM)

White‐Mathieu,

Baskin

2024

Current Protocols

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Fluorescent imaging of cellular membranes is challenged by the size of lipid bilayers, which are smaller than the diffraction limit of light. Recently, expansion microscopy (ExM) has emerged as an approachable super‐resolution method that requires only widely accessible confocal microscopes. In this method, biomolecules of interest are anchored to hydrogel‐based, polymeric networks that are expanded through osmosis to physically separate and resolve features smaller than the diffraction limit of light. Whereas ExM has been employed for super‐resolution imaging of proteins, DNA, RNA, and glycans, the application of this method to the study of lipids is challenged by the requirement of permeabilization procedures that remove lipids and compromise the integrity of the membrane. Here, we describe our recently developed protocols for lipid expansion microscopy (LExM), a method that enables ExM of membranes without permeabilization. These detailed protocols and accompanying commentary sections aim to make LExM accessible to any experimentalist interested in imaging membranes with super‐resolution. © 2024 Wiley Periodicals LLC.Basic Protocol 1: LExM of alkyne‐choline lipidsBasic Protocol 2: LExM of IMPACT‐labeled lipidsBasic Protocol 3: LExM of clickable cholesterolBasic Protocol 4: Determining the expansion factor

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Expansion microscopy: A chemical approach for super-resolution microscopy

Cited by 18 publications

References 65 publications

Deployment of the natural magnifier CMTKG for naked expansion microscopy of biological tissues

Deployment of the natural magnifier CMTKG for naked expansion microscopy of biological tissues

Bio-orthogonal Glycan Imaging of Culture Cells and Whole AnimalC. eleganswith Expansion Microscopy

Super‐Resolution Imaging of Clickable Lipids With Lipid Expansion Microscopy (LExM)

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