Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy
Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro prob...
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Bio-protocol LLC
2025-01-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5150&type=0 |
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| author | Xichuan Ge Wei Ren Chunyan Shan Peng Xi Baoxiang Gao |
| author_facet | Xichuan Ge Wei Ren Chunyan Shan Peng Xi Baoxiang Gao |
| author_sort | Xichuan Ge |
| collection | DOAJ |
| description | Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized. |
| format | Article |
| id | doaj-art-a6a406af3efd498cbad7e85f71635355 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-a6a406af3efd498cbad7e85f716353552025-02-07T08:16:31ZengBio-protocol LLCBio-Protocol2331-83252025-01-0115110.21769/BioProtoc.5150Using HBmito Crimson to Observe Mitochondrial Cristae Through STED MicroscopyXichuan Ge0Wei Ren1Chunyan Shan2Peng Xi3Baoxiang Gao4Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Material Science, Hebei University. Baoding, ChinaAiry Technologies Co., Ltd., Beijing, ChinaDepartment of Biomedical Engineering, College of Future Technology, Peking University, Beijing, ChinaNational Biomedical Imaging Center, Peking University, Beijing, ChinaSchool of Life Sciences, Peking University, Beijing, ChinaNational Center for Protein Sciences, Peking University, Beijing, ChinaDepartment of Biomedical Engineering, College of Future Technology, Peking University, Beijing, ChinaNational Biomedical Imaging Center, Peking University, Beijing, ChinaKey Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Material Science, Hebei University. Baoding, ChinaMitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized.https://bio-protocol.org/en/bpdetail?id=5150&type=0 |
| spellingShingle | Xichuan Ge Wei Ren Chunyan Shan Peng Xi Baoxiang Gao Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy Bio-Protocol |
| title | Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy |
| title_full | Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy |
| title_fullStr | Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy |
| title_full_unstemmed | Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy |
| title_short | Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy |
| title_sort | using hbmito crimson to observe mitochondrial cristae through sted microscopy |
| url | https://bio-protocol.org/en/bpdetail?id=5150&type=0 |
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