Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging
Abstract Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-48959-9 |
| _version_ | 1797388316467789824 |
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| author | Ueh-Ting Tim Wang Xuejiao Tian Yae-Huei Liou Sue-Ping Lee Hsiao-Tang Hu Chieh-Han Lu Po-Ting Lin Ya-Jen Cheng Peilin Chen Bi-Chang Chen |
| author_facet | Ueh-Ting Tim Wang Xuejiao Tian Yae-Huei Liou Sue-Ping Lee Hsiao-Tang Hu Chieh-Han Lu Po-Ting Lin Ya-Jen Cheng Peilin Chen Bi-Chang Chen |
| author_sort | Ueh-Ting Tim Wang |
| collection | DOAJ |
| description | Abstract Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy. |
| first_indexed | 2024-03-08T22:39:00Z |
| format | Article |
| id | doaj.art-08f6e3c338d84483ad4ed15e0ae3db05 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-08T22:39:00Z |
| publishDate | 2023-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-08f6e3c338d84483ad4ed15e0ae3db052023-12-17T12:15:32ZengNature PortfolioScientific Reports2045-23222023-12-0113111510.1038/s41598-023-48959-9Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imagingUeh-Ting Tim Wang0Xuejiao Tian1Yae-Huei Liou2Sue-Ping Lee3Hsiao-Tang Hu4Chieh-Han Lu5Po-Ting Lin6Ya-Jen Cheng7Peilin Chen8Bi-Chang Chen9Affiliated Senior High School of National Taiwan Normal UniversityResearch Center for Applied Sciences, Academia SinicaInstitute of Molecular Biology, Academia SinicaInstitute of Molecular Biology, Academia SinicaInstitute of Molecular Biology, Academia SinicaResearch Center for Applied Sciences, Academia SinicaResearch Center for Applied Sciences, Academia SinicaInstitute of Molecular Biology, Academia SinicaResearch Center for Applied Sciences, Academia SinicaResearch Center for Applied Sciences, Academia SinicaAbstract Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.https://doi.org/10.1038/s41598-023-48959-9 |
| spellingShingle | Ueh-Ting Tim Wang Xuejiao Tian Yae-Huei Liou Sue-Ping Lee Hsiao-Tang Hu Chieh-Han Lu Po-Ting Lin Ya-Jen Cheng Peilin Chen Bi-Chang Chen Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging Scientific Reports |
| title | Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging |
| title_full | Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging |
| title_fullStr | Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging |
| title_full_unstemmed | Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging |
| title_short | Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging |
| title_sort | protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3d imaging |
| url | https://doi.org/10.1038/s41598-023-48959-9 |
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