Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin
Expansion microscopy (ExM) is an imaging technique that enables super-resolution imaging of biological specimens using conventional confocal microscopy. This process entails the isotropic physical expansion of a (biomolecular) sample that has been cross-linked to a swellable polymer. The grafting of...
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Bio-protocol LLC
2025-04-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5273&type=0 |
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| author | Jianjun Huang Gang Wen Thibo Iven Débora Linhares Leewon Koo Markus Sauer Wim Dehaen Volker Leen Johan Hofkens |
| author_facet | Jianjun Huang Gang Wen Thibo Iven Débora Linhares Leewon Koo Markus Sauer Wim Dehaen Volker Leen Johan Hofkens |
| author_sort | Jianjun Huang |
| collection | DOAJ |
| description | Expansion microscopy (ExM) is an imaging technique that enables super-resolution imaging of biological specimens using conventional confocal microscopy. This process entails the isotropic physical expansion of a (biomolecular) sample that has been cross-linked to a swellable polymer. The grafting of biomolecules (and the subsequent fluorescent readout) is accomplished by introducing an acryloyl group to the amine groups of lysine residues within the proteins, enabling subsequent imaging. However, visualizing actin filaments with high spatial resolution using ExM remains challenging. Herein, we report the construction of a phalloidin conjugate containing actin stains and their application in ExM. This protocol highlights the efficacy of trifunctional linker (TRITON/Actin-ExM) for F-actin imaging, demonstrating that TRITON-labeled actin allows for efficient anchoring and signal retention, enabling robust visualization of actin filaments in expansion microscopy. |
| format | Article |
| id | doaj-art-61c7ec15f8e745ecb76b359364bcd900 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-61c7ec15f8e745ecb76b359364bcd9002025-08-20T03:48:46ZengBio-protocol LLCBio-Protocol2331-83252025-04-0115810.21769/BioProtoc.5273Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated PhalloidinJianjun Huang0Gang Wen1Thibo Iven2Débora Linhares3Leewon Koo4Markus Sauer5Wim Dehaen6Volker Leen7Johan Hofkens8Department of Chemistry, KU Leuven, Leuven, BelgiumDepartment of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, Würzburg, GermanyDepartment of Chemistry, KU Leuven, Leuven, BelgiumDepartment of Chemistry, KU Leuven, Leuven, BelgiumDepartment of Chemistry, KU Leuven, Leuven, BelgiumDepartment of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, Würzburg, GermanyDepartment of Chemistry, KU Leuven, Leuven, BelgiumChrometra Scientific, Kortenaken, BelgiumDepartment of Chemistry, KU Leuven, Leuven, BelgiumMax Planck Institute for Polymer Research, Mainz, GermanyExpansion microscopy (ExM) is an imaging technique that enables super-resolution imaging of biological specimens using conventional confocal microscopy. This process entails the isotropic physical expansion of a (biomolecular) sample that has been cross-linked to a swellable polymer. The grafting of biomolecules (and the subsequent fluorescent readout) is accomplished by introducing an acryloyl group to the amine groups of lysine residues within the proteins, enabling subsequent imaging. However, visualizing actin filaments with high spatial resolution using ExM remains challenging. Herein, we report the construction of a phalloidin conjugate containing actin stains and their application in ExM. This protocol highlights the efficacy of trifunctional linker (TRITON/Actin-ExM) for F-actin imaging, demonstrating that TRITON-labeled actin allows for efficient anchoring and signal retention, enabling robust visualization of actin filaments in expansion microscopy.https://bio-protocol.org/en/bpdetail?id=5273&type=0 |
| spellingShingle | Jianjun Huang Gang Wen Thibo Iven Débora Linhares Leewon Koo Markus Sauer Wim Dehaen Volker Leen Johan Hofkens Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin Bio-Protocol |
| title | Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin |
| title_full | Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin |
| title_fullStr | Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin |
| title_full_unstemmed | Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin |
| title_short | Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin |
| title_sort | visualization of f actin through expansion microscopy exm with trifunctional linker conjugated phalloidin |
| url | https://bio-protocol.org/en/bpdetail?id=5273&type=0 |
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