Evaluation of Direct Grafting Strategies <i>via</i> Trivalent Anchoring for Enabling Lipid Membrane and Cytoskeleton Staining in Expansion Microscopy

Wen, Gang; Vanheusden, Marisa; Acke, Aline; Valli, Donato; Neely, Robert K.; Leen, Volker; Hofkens, Johan

doi:10.1021/acsnano.9b09259

Cited by 77 publications

(115 citation statements)

References 26 publications

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“…8 and 9) 34,35 our findings are highly relevant. Very recently 36,37 , trifunctional linkers have been introduced that are inert to polymerization, digestion and denaturation, and enable direct covalent linking of target molecules and functional groups to the hydrogel. Therefore, trifunctional linkers can retain a high number of labels and fluorescence molecules available for postexpansion imaging.…”

Section: Discussionmentioning

confidence: 99%

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

et al. 2020

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Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with singlemolecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

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Section: Discussionmentioning

confidence: 99%

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

et al. 2020

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“…Compared with these label amplification methods, LR-ExM not only avoids the polymerization-induced fluorophore damage by post-expansion labeling, but eliminates the digestion-induced fluorophore loss by directly crosslinking the reporter to the hydrogel. Wen and coworkers presented a similar chemical linking approach that enables direct grafting of a targeting molecule and fluorophore to the hydrogel, and demonstrated the design principle of trifunctional anchors also work for antibody-free staining of small biomolecules like lipid and actin in ExM ( 42 ).…”

Section: Discussionmentioning

confidence: 99%

“…ExFISH ( 15 )), lipid (e.g. mExM( 45 ), TRITON( 42 )), context protein (e.g. FLARE( 46 ), pan-ExM( 7 )), and so on.…”

Section: Discussionmentioning

confidence: 99%

Label-retention expansion microscopy

Shi

Dai

et al. 2019

Preprint

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Expansion microscopy (ExM) improves the resolution of fluorescence microscopy by physically expanding the sample embedded in a hydrogel 1-4 . Since its invention, ExM has been successfully applied to a wide range of cell, tissue and animal samples 2-9 . Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency protein labeling using enzymatic tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with super-resolution Stochastic Optical Reconstruction Microscopy (STORM), we have achieved 5 nm resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.By physically expanding the sample before image acquisition, ExM has enabled the use of a conventional confocal microscope to achieve ~ 70 nm lateral spatial resolution 2-4, 6, 9, 10 . Recent efforts have further enhanced the resolution of ExM either by increasing the volume expansion ratio 11,12 or by combining ExM with super-resolution microscopy such as Structured Illumination Microscopy (SIM) 5,8,13 and Stimulated Emission Depletion (STED) microscopy 7,14,15 . In all these cases, the homogenization of the specimen through either protease digestion 10 or protein denaturation 2, 3 is essential to achieve isotropic expansion without structural distortion. To retain the spatial information of the target structures, the biomolecules of interest (e.g. protein 2-4 , RNA 1 ) and/or labels (e.g. dye-labeled DNA 10 , dye-labeled antibodies 2, 4 or fluorescent proteins 4 ) are anchored to the hydrogel matrix prior to digestion or denaturation. Nevertheless, digestion and denaturation cause incompletely anchored proteins or protein fragments to be washed out, the polymerization reaction to make the hydrogel produces free radicals that readily destroy fluorophores, and both factors can damage fluorescent proteins. Consequently, more than 50% of the target molecules can lose labeling after expansion 4 , which is a major issue of current ExM methods 16,17 . This label loss is exacerbated when aiming for higher spatial resolution. First, ensuring isotropic expansion at nanometer scale requires more thorough digestion or denaturation, hence more wash-out. Second, superresolution microscopy often requires certain dyes that do not survive hydrogel polymerization reaction. For example, Alexa Fluor (AF) 647, one of the best fluorophores for STORM, suffers > 90% loss of the fluorescent molecules after polymerization and

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“…Furthermore, a biotin residue is attached to enable fluorescence staining of the probe with labeled streptavidin. Alternatively, a trifunctional linker strategy termed TRITON has been introduced for ExM that enables simultaneous targeting, labeling, and grafting of biomolecules using a monomer unit (acryloyl) into the hydrogel 26 . Using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) as small-molecule targeting staining phospholipid bilayers, cellular membranes can be expanded and imaged.…”

Section: Resultsmentioning

confidence: 99%

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

et al. 2020

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Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

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Evaluation of Direct Grafting Strategies via Trivalent Anchoring for Enabling Lipid Membrane and Cytoskeleton Staining in Expansion Microscopy

Cited by 77 publications

References 26 publications

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Label-retention expansion microscopy

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

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