Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization

Cainero, Isotta; Oneto, Michele; Pesce, Luca; Zanini, Giulia; Lanzanò, Luca; Diaspro, Alberto; Bianchini, Paolo

doi:10.1016/j.bpj.2016.11.775

Cited by 6 publications

(7 citation statements)

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“…Additionally, similar improvements in performance may be obtainable by combining specimen expansion with other super-resolution modalities including SMLM and STED. 20,48 …”

Section: Discussionmentioning

confidence: 99%

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

et al. 2017

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Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ~65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ~30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 which localizes to disc microribbons, and the functionally unknown DAP16263 which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.

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“…Additionally, similar improvements in performance may be obtainable by combining specimen expansion with other super-resolution modalities including SMLM and STED. 20,48 …”

Section: Discussionmentioning

confidence: 99%

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

et al. 2017

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“…Such a resolution is typical for super‐resolution techniques like stimulated emission depletion (STED) , saturated structured illumination (SSIM) , and photoactivated localization microscopy (PALM) . Since ExM generally preserves the fluorescence, we can apply a super‐resolved method like STED to the expanded sample achieving a further notable increase in spatial resolution . The immediate result using STED is that an additional resolution gain of 2‐ to 3‐fold can be reached at low depletion power, also suggesting the parallelization of the process can be achieved towards high throughput imaging .…”

Section: Introductionmentioning

confidence: 99%

“…Indeed, the combination of ExM with other super‐resolution methods, for example, ExM‐STED (ExSTED) , UltraExM STED , and SIM‐ExM , has been recently used to show the isotropic expansion for cytoplasmic structures, such as the cytoskeleton, and symmetric macromolecular complexes like the centrosomes , and centrioles . Such solutions enabled a resolution of about 10 nm , suggesting its relevance to quantify the isotropic expansion at the nanoscale level. However, the overall fluorescence photon budget can be reduced by recurring and high illumination imposing a new strategy.…”

Section: Introductionmentioning

confidence: 99%

Measuring expansion from macro‐ to nanoscale using NPC as intrinsic reporter

Pesce

Cozzolino

Lanzanò

et al. 2019

Journal of Biophotonics

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Expansion microscopy is a super‐resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. One of the main concerns in this approach regards the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well‐known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale to measure subcellular features with an accuracy from 10 to 5 nm. Therefore, it is an excellent method for structural studies of macromolecular complexes.

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“…Such a resolution is typical for super-resolution techniques like STimulated Emission Depletion (STED) ( 5-7 ), Saturated Structured Illumination (SSIM) ( 8 ), and Photoactivated Localization Microscopy (PALM) ( 9 ). Since ExM generally preserves the fluorescence, we can apply a super-resolved method like STED to the expanded sample achieving a further notable increase in spatial resolution ( 10, 11 ). The immediate result using STED is that an additional resolution gain of 2-3 fold can be reached at low depletion power and reduced photobleaching rate, also suggesting the parallelization of the process can be achieved towards high throughput imaging ( 12 ).…”

Section: Introductionmentioning

confidence: 99%

“…ExM-STED (ExSTED) ( 11 ) and SIM-ExM ( 13 ), has been recently used to show the isotropic expansion for cytoplasmic structures, such as the cytoskeleton, and symmetric macromolecular complexes like the centrosomes ( 11 ). In particular, ExSTED enabled a resolution of about 10 nm ( 10, 11 ), suggesting its relevance to quantify the isotropic expansion at the nanoscale level. However, the overall fluorescence photon budget can be reduced by strong illumination imposing a new strategy.…”

Section: Introductionmentioning

confidence: 99%

Enigma at the nanoscale: can the NPC act as an intrinsic reporter for isotropic expansion microscopy?

Pesce

Cozzolino

Lanzanò

et al. 2018

Preprint

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13Expansion microscopy is a super-resolution method that allows expanding uniformly biological samples, by 14increasing the relative distances among fluorescent molecules labeling specific components. The main "enigma" 15 regarding this approach is given by the isotropic behavior at the nanoscale. The present study aims to determine the 16 robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus 17is on the nuclear pore complex (NPC), as well-known nanoscale component endowed of a preserved and symmetrical 18 structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address 19 the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, 20allows concluding that expansion microscopy can be used at the nanoscale with a uniform accuracy in the range of 20 21nm. In addition, it is an excellent method for structural studies of macromolecular complexes. 22 23 24

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Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization

Abstract: High-density lipoprotein (HDL) is involved in the transport and metabolism of phospholipids, triglycerides, and cholesterol. Mimics of HDL are being explored as potential therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized

Cited by 6 publications

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Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

Measuring expansion from macro‐ to nanoscale using NPC as intrinsic reporter

Enigma at the nanoscale: can the NPC act as an intrinsic reporter for isotropic expansion microscopy?

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