Enhanced expansion microscopy to measure nanoscale structural and biochemical remodeling in single cells

Sheard, Thomas M. D.; Jayasinghe, Izzy

doi:10.1016/bs.mcb.2020.04.019

Cited by 9 publications

(15 citation statements)

References 48 publications

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“…Since expansion microscopy is a sample preparation method, it is not limited to the imaging approach, and it can be combined with standard CLSM or ACLSM ( Sheard and Jayasinghe, 2021 ) but also with super-resolution regimes such as single-molecule localization approaches with STORM ( Xu et al, 2019 ) as well as with STED ( Gao et al, 2018 , 2021 ).…”

Section: Expansion Microscopymentioning

confidence: 99%

Imaging plant cells and organs with light-sheet and super-resolution microscopy

et al. 2021

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The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.

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Section: Expansion Microscopymentioning

confidence: 99%

Imaging plant cells and organs with light-sheet and super-resolution microscopy

et al. 2021

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“…EExM, despite the far superior 3D resolution that it offers in resolving both the topologies of the dyads and individual RyRs, still requires a number of calibrations and tests. These include independent estimates of the gel expansion factors and isotropy of expansion (see detailed discussion in (23)). Combining the X10 expansion protocol with imaging modalities such as STED or SIM can further improve the resolution attainable to a sub-10-nm regime.…”

Section: Bottlenecks and Motivations For Expansion Microscopy Of Cardiac Ryr Clustersmentioning

confidence: 99%

“…The major axes of the gel were measured to calculate the pre-expansion size. We also performed 4x EExM on tissue sections, similar to that described for isolated cardiomyocytes previously (23). The gel solution, according to the proExM protocol previously described (26), was made in advance and defrosted from frozen aliquots.…”

Section: Expansion Microscopymentioning

confidence: 99%

“…First, samples were incubated with 0.1 mg/ml acryloyl-X (Thermo Fisher Scientific) in PBS overnight at 4°C, then washed in PBS immediately prior to addition of gel solution. We performed 10x EExM on isolated cardiomyocytes as detailed previously (5,23). X10 gels (4:1 molar ratio of dimethylacrylamide (Sigma-Aldrich) and sodium acrylate (Sigma-Aldrich), dissolved in deionized H20 (dH2O)) were made according to the previous recipe (24,25).…”

Section: Expansion Microscopymentioning

confidence: 99%

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Molecular-scale 3D visualisation of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads

Sheard

Hurley

Smith

et al. 2021

Preprint

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Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery in cardiomyocytes. Various adaptations of super-resolution microscopy have revealed intricate details of the structure, molecular composition and locations of these couplons. However, most optical super-resolution techniques lack the capacity for three-dimensional (3D) visualisation. Enhanced Expansion Microscopy (EExM) offers resolution (in-plane and axially) sufficient to spatially resolve individual proteins within peripheral couplons and within dyads located in the interior. We have combined immunocytochemistry and immunohistochemistry variations of EExM with 3D visualisation to examine the complex topologies, geometries and molecular sub-domains within RyR clusters. We observed that peripheral couplons exhibit variable co-clustering ratios and patterns between RyR and the structural protein, junctophilin-2 (JPH2). Dyads possessed sub-domains of JPH2 which occupied the central regions of the RyR cluster, whilst the poles were typically devoid of JPH2 and broader, and likely specialise in turnover and remodelling of the cluster. In right ventricular myocytes from rats with monocrotaline-induced right ventricular failure, we observed hallmarks of RyR cluster fragmentation accompanied by similar fragmentations of the JPH2 sub-domains. We hypothesise that the frayed morphology of RyRs in close proximity to fragmented JPH2 structural sub-domains may form the primordial foci of RyR mobilisation and dyad remodelling.

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“…To improve upon the resolution achievable with standard proExM in conjunction with confocal microscopy, we used the resolution-doubling Airyscan protocol (called Enhanced Expansion Microscopy, EExM; (Sheard and Jayasinghe, 2021)) to image the proExM gels containing pan-protein staining of the bulk proteome in dissected C. elegans germlines. Among other intracellular features, we observed P granules at nanoscale resolution (∼40nm in plane and ∼100nm axial).…”

Section: Introductionmentioning

confidence: 99%

Expansion microscopy reveals subdomains in C. elegans germ granules

Suen

Sheard

Lin

et al. 2022

Preprint

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Many membraneless organelles (MLOs) have been shown to form via liquid-liquid phase separation (LLPS). Light and electron microscopy techniques have been indispensable in the identification and characterization of LLPS MLOs. However, for complex MLOs such as the perinuclear germ granule in C. elegans, our understanding of how the organelle as a whole is regulated is hampered by 1) the technical limitations in confocal fluorescence imaging in which only a few granule protein markers can be examined at a time and 2) the inaccessibility of electron microscopy. In this study, we take advantage of the newly developed super-resolution method of expansion microscopy (ExM) and in-situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with wild type animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. The experimental workflow developed here will serve as an important tool in our understanding of germ granule biology as well as the biological role of LLPS.

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Enhanced expansion microscopy to measure nanoscale structural and biochemical remodeling in single cells

Cited by 9 publications

References 48 publications

Imaging plant cells and organs with light-sheet and super-resolution microscopy

Imaging plant cells and organs with light-sheet and super-resolution microscopy

Molecular-scale 3D visualisation of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads

Expansion microscopy reveals subdomains in C. elegans germ granules

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