Label-retention expansion microscopy

Shi, Xiaoyu; Li, Qi; Dai, Zhipeng; Tran, Arthur A; Feng, Siyu; Ramirez, Alexander; Lin, Zixi; Chow, Tracy T.; Chen, Jiapei; Kumar, Dhivya; McColloch, Andrew; Reiter, Jeremy F.; Huang, Eric J.; Seiple, Ian B.

doi:10.1083/jcb.202105067

Cited by 45 publications

(56 citation statements)

References 48 publications

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“…[ 228 ] Alternatively, the effective probe size can be reduced by physically swelling the sample volume. [ 229 , 230 , 231 ] By this method, the linkage error of a pair of primary and secondary antibodies can be reduced from 15 to 7 nm after expansion. [ 229 ] Likewise, large photostable probes such as nanoparticles and encapsulated dyes can have sub‐10‐nm linkage error after expansion.…”

Section: Discussionmentioning

confidence: 99%

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

2022

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Photobleaching is the permanent loss of fluorescence after extended exposure to light and is a major limiting factor in super‐resolution microscopy (SRM) that restricts spatiotemporal resolution and observation time. Strategies for preventing or overcoming photobleaching in SRM are reviewed developing new probes and chemical environments. Photostabilization strategies are introduced first, which are borrowed from conventional fluorescence microscopy, that are employed in SRM. SRM‐specific strategies are then highlighted that exploit the on–off transitions of fluorescence, which is the key mechanism for achieving super‐resolution, which are becoming new routes to address photobleaching in SRM. Off states can serve as a shelter from excitation by light or an exit to release a damaged probe and replace it with a fresh one. Such efforts in overcoming the photobleaching limits are anticipated to enhance resolution to molecular scales and to extend the observation time to physiological lifespans.

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Section: Discussionmentioning

confidence: 99%

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

2022

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Section: Resolution and Expansion Isotropymentioning

confidence: 99%

Nanoscale fluorescence imaging of biological ultrastructure via molecular anchoring and physical expansion

et al. 2022

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Nanoscale imaging of biological samples can provide rich morphological and mechanistic information about biological functions and dysfunctions at the subcellular and molecular level. Expansion microscopy (ExM) is a recently developed nanoscale fluorescence imaging method that takes advantage of physical enlargement of biological samples. In ExM, preserved cells and tissues are embedded in a swellable hydrogel, to which the molecules and fluorescent tags in the samples are anchored. When the hydrogel swells several-fold, the effective resolution of the sample images can be improved accordingly via physical separation of the retained molecules and fluorescent tags. In this review, we focus on the early conception and development of ExM from a biochemical and materials perspective. We first examine the general workflow as well as the numerous variations of ExM developed to retain and visualize a broad range of biomolecules, such as proteins, nucleic acids, and membranous structures. We then describe a number of inherent challenges facing ExM, including those associated with expansion isotropy and labeling density, as well as the ongoing effort to address these limitations. Finally, we discuss the prospect and possibility of pushing the resolution and accuracy of ExM to the single-molecule scale and beyond.

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“…In principle, a simple procedure for further enhancing resolution would be the combination of ExM with conventional super-resolution techniques. This has been attempted many times, relying typically on the original 4x protocol of the Boyden laboratory, using, for example, stimulated emission depletion microscopy (STED) [12][13][14], stochastic optical reconstruction microscopy (STORM) [15][16][17], or structured illumination microscopy (SIM) [18][19][20]. However, the resulting imaging precision is often only slightly higher than that obtained in optimal implementations of STORM microscopy [21,22].…”

Section: Introductionmentioning

confidence: 99%

“…While some of the ExM-optical superresolution protocols reached resolutions of 4-8 nm in individual imaging channels, using either STED or STORM (e.g. [12,17]), the use of 4x ExM seems to limit imaging precision, requiring the use of gels with larger expansion factors.…”

Section: Introductionmentioning

confidence: 99%

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Saal

Shaib

Mougios

et al. 2022

Preprint

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Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the 10-fold expansion of the samples through high-temperature homogenization (X10ht). The resulting gels exhibited relatively high fluorescence intensity, enabling the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm. X10ht offers a more thorough homogenization than previous X10 protocols based on enzymatic digestion, and thereby enables the expansion of thick samples. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

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Label-retention expansion microscopy

Cited by 45 publications

References 48 publications

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

Nanoscale fluorescence imaging of biological ultrastructure via molecular anchoring and physical expansion

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

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