Expansion Stimulated Emission Depletion Microscopy (ExSTED)

Gao, Mengfei; Maraspini, Riccardo; Beutel, Oliver; Zehtabian, Amin; Eickholt, Britta J.; Honigmann, Alf; Ewers, Helge

doi:10.1021/acsnano.8b00776

Cited by 160 publications

(156 citation statements)

References 41 publications

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“…To further enhance the resolution, ExM has been combined with structured illumination microscopy (SIM) 13,14 and stimulated emission depletion (STED) microscopy 2,15 . By careful optimization of the expansion protocol U-ExM demonstrated that even ultrastructural details of multiprotein complexes such as centrioles can be truthfully preserved 2 .…”

Section: Introductionmentioning

confidence: 99%

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

Zwettler¹,

Reinhard²,

Gambarotto

et al. 2020

Preprint

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Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule 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: Introductionmentioning

confidence: 99%

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

Zwettler¹,

Reinhard²,

Gambarotto

et al. 2020

Preprint

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“…In principle, super‐resolution instruments can be used with ExM samples to harness the advantages of both methods. Structured illumination microscopy (SIM), for example, was applied to expanded samples to achieve resolutions comparable with those of SMLM, whereas STED combined with ExM (ExSTED) produced a resolution of 10 nm . However, ExSTED cannot utilize the highest STED resolution because photobleaching prevents the use of the maximum depletion intensity.…”

Section: Methodsmentioning

confidence: 99%

Amplified Expansion Stimulated Emission Depletion Microscopy

Kim

Lee

et al. 2019

ChemBioChem

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Expansion microscopy (ExM) enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of ≈4 in one dimension and a factor of ≈64 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10 nm. However, photobleaching is a critical issue in ExSTED because the sample expansion lowers the fluorophore density whereas high‐resolution STED requires high depletion intensity. To overcome these issues, we developed extremely bright expansion nanoscopy by using biotin–avidin signal amplification to increase the labeling density. Our method provides up to sevenfold increases in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with maximum depletion intensities of a commercial microscope in the order of GW cm−2. We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle.

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“…CRL-1651) that are easy to culture and grow rapidly (Gluzman, 1981), but other mammalian cell lines are also typically used such as U2OS , BS-C-1 (Bates et al, 2007;Lin et al, 2018), or HeLa, which are thicker and can be more challenging to image (Gao et al, 2018). It is important to know a bit about the underlying biology of the chosen cell line (use as a biological model, tumor origin…) in order to understand the possible singularities in their organization.…”

Section: Sample Preparationmentioning

confidence: 99%

“…Moreover, higher labeling density for a given structure can be obtained by using several antibodies against different epitopes within this structure. This strategy has been used to maximize signal for synapses (Sigal et al, 2015) and microtubules in SMLM or Expansion Microscopy (Gao et al, 2018). Similarly, we usually label microtubules with a combination of anti-αand anti-ß-tubulin monoclonal antibodies to obtain a dense labeling in STORM and PAINT ( Fig.…”

Section: Blocking and Primary Antibodies Incubationmentioning

confidence: 99%

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Jimenez

Friedl

Leterrier

2019

Preprint

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Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

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Expansion Stimulated Emission Depletion Microscopy (ExSTED)

Cited by 160 publications

References 41 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)

Amplified Expansion Stimulated Emission Depletion Microscopy

About samples, giving examples: Optimized Single Molecule Localization Microscopy

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