Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites
Direct stochastic optical reconstruction microscopy (dSTORM) circumvents the diffraction limit of light, emerging as a powerful superresolution technique for visualizing subcellular structures with a nanoscale resolution of 10–20 nm. Yet achieving ultrastructural resolution using dSTORM alone remain...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-06-01
|
| Series: | Gels |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2310-2861/11/7/491 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849246564212539392 |
|---|---|
| author | Jimmy Ching-Cheng Hsu T. Tony Yang |
| author_facet | Jimmy Ching-Cheng Hsu T. Tony Yang |
| author_sort | Jimmy Ching-Cheng Hsu |
| collection | DOAJ |
| description | Direct stochastic optical reconstruction microscopy (dSTORM) circumvents the diffraction limit of light, emerging as a powerful superresolution technique for visualizing subcellular structures with a nanoscale resolution of 10–20 nm. Yet achieving ultrastructural resolution using dSTORM alone remains challenging, despite its advantage of requiring only minimal modifications to the imaging setup and sample preparation compared to conventional fluorescence microscopy. A recent advancement that integrates expansion microscopy (ExM), which embeds specimens in a swellable polymer gel, with dSTORM holds promise for attaining imaging resolutions below 10 nm. The combined resolution, however, is governed by the expansion factor of samples, and prior studies have primarily focused on integrations involving approximately 4-fold gel expansion, as dSTORM imaging of high-fold-expanded specimens is still technically demanding. Here, we propose a pragmatic expansion strategy—post-labeling ten-fold robust expansion microscopy (plTREx)—and outline a workflow to facilitate its compatibility with dSTORM, collectively termed plTREx-dSTORM. Specifically, this workflow enhances the mechanical stability of the expansion hydrogel and improves fluorescence signal density across both widefield and dSTORM imaging platforms. Furthermore, we optimize the re-embedding protocol to integrate hydrogel expansion with dSTORM while preventing gel shrinkage. Together, plTREx-dSTORM enables highly refined imaging capable of ultrastructural interpretation of cellular proteins, effectively bridging the resolution gap between electron microscopy and optical microscopy. |
| format | Article |
| id | doaj-art-7d88c138cf7547dc8892906ff59d333a |
| institution | Kabale University |
| issn | 2310-2861 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Gels |
| spelling | doaj-art-7d88c138cf7547dc8892906ff59d333a2025-08-20T03:58:27ZengMDPI AGGels2310-28612025-06-0111749110.3390/gels11070491Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel CompositesJimmy Ching-Cheng Hsu0T. Tony Yang1Department of Electrical Engineering, National Taiwan University, Taipei 10617, TaiwanDepartment of Electrical Engineering, National Taiwan University, Taipei 10617, TaiwanDirect stochastic optical reconstruction microscopy (dSTORM) circumvents the diffraction limit of light, emerging as a powerful superresolution technique for visualizing subcellular structures with a nanoscale resolution of 10–20 nm. Yet achieving ultrastructural resolution using dSTORM alone remains challenging, despite its advantage of requiring only minimal modifications to the imaging setup and sample preparation compared to conventional fluorescence microscopy. A recent advancement that integrates expansion microscopy (ExM), which embeds specimens in a swellable polymer gel, with dSTORM holds promise for attaining imaging resolutions below 10 nm. The combined resolution, however, is governed by the expansion factor of samples, and prior studies have primarily focused on integrations involving approximately 4-fold gel expansion, as dSTORM imaging of high-fold-expanded specimens is still technically demanding. Here, we propose a pragmatic expansion strategy—post-labeling ten-fold robust expansion microscopy (plTREx)—and outline a workflow to facilitate its compatibility with dSTORM, collectively termed plTREx-dSTORM. Specifically, this workflow enhances the mechanical stability of the expansion hydrogel and improves fluorescence signal density across both widefield and dSTORM imaging platforms. Furthermore, we optimize the re-embedding protocol to integrate hydrogel expansion with dSTORM while preventing gel shrinkage. Together, plTREx-dSTORM enables highly refined imaging capable of ultrastructural interpretation of cellular proteins, effectively bridging the resolution gap between electron microscopy and optical microscopy.https://www.mdpi.com/2310-2861/11/7/491superresolution microscopySMLMdSTORMexpansion microscopy (ExM) |
| spellingShingle | Jimmy Ching-Cheng Hsu T. Tony Yang Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites Gels superresolution microscopy SMLM dSTORM expansion microscopy (ExM) |
| title | Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites |
| title_full | Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites |
| title_fullStr | Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites |
| title_full_unstemmed | Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites |
| title_short | Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites |
| title_sort | pushing optical resolution to the few nanometer scale via dstorm imaging of expanded specimen gel composites |
| topic | superresolution microscopy SMLM dSTORM expansion microscopy (ExM) |
| url | https://www.mdpi.com/2310-2861/11/7/491 |
| work_keys_str_mv | AT jimmychingchenghsu pushingopticalresolutiontothefewnanometerscaleviadstormimagingofexpandedspecimengelcomposites AT ttonyyang pushingopticalresolutiontothefewnanometerscaleviadstormimagingofexpandedspecimengelcomposites |