Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing
Abstract Multicolor microscopy and super-resolution optical microscopy are two widely used techniques that greatly enhance the ability to distinguish and resolve structures in cellular imaging. These methods have individually transformed cellular imaging by allowing detailed visualization of cellula...
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| Format: | Article |
| Language: | English |
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Nature Publishing Group
2025-01-01
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| Series: | Light: Science & Applications |
| Online Access: | https://doi.org/10.1038/s41377-024-01711-y |
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| author | Luwei Wang Yue Chen Jiaqing Guo Xiaoyu Weng Wei Yan Jun Song Tong Ye Junle Qu |
| author_facet | Luwei Wang Yue Chen Jiaqing Guo Xiaoyu Weng Wei Yan Jun Song Tong Ye Junle Qu |
| author_sort | Luwei Wang |
| collection | DOAJ |
| description | Abstract Multicolor microscopy and super-resolution optical microscopy are two widely used techniques that greatly enhance the ability to distinguish and resolve structures in cellular imaging. These methods have individually transformed cellular imaging by allowing detailed visualization of cellular and subcellular structures, as well as organelle interactions. However, integrating multicolor and super-resolution microscopy into a single method remains challenging due to issues like spectral overlap, crosstalk, photobleaching, phototoxicity, and technical complexity. These challenges arise from the conflicting requirements of using different fluorophores for multicolor labeling and fluorophores with specific properties for super-resolution imaging. We propose a novel multicolor super-resolution imaging method called phasor-based fluorescence spatiotemporal modulation (Phasor-FSTM). This method uses time-resolved detection to acquire spatiotemporal data from encoded photons, employs phasor analysis to simultaneously separate multiple components, and applies fluorescence modulation to create super-resolution images. Phasor-FSTM enables the identification of multiple structural components with greater spatial accuracy on an enhanced laser scanning confocal microscope using a single-wavelength laser. To demonstrate the capabilities of Phasor-FSTM, we performed two-color to four-color super-resolution imaging at a resolution of ~λ/5 and observed the interactions of organelles in live cells during continuous imaging for a duration of over 20 min. Our method stands out for its simplicity and adaptability, seamlessly fitting into existing laser scanning microscopes without requiring multiple laser lines for excitation, which also provides a new avenue for other super-resolution imaging technologies based on different principles to build multi-color imaging systems with the requirement of a lower budget. |
| format | Article |
| id | doaj-art-6d510f81ece14c958ca5aecfbf4de5fa |
| institution | Kabale University |
| issn | 2047-7538 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Publishing Group |
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| series | Light: Science & Applications |
| spelling | doaj-art-6d510f81ece14c958ca5aecfbf4de5fa2025-01-05T12:46:55ZengNature Publishing GroupLight: Science & Applications2047-75382025-01-0114111310.1038/s41377-024-01711-yPhasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexingLuwei Wang0Yue Chen1Jiaqing Guo2Xiaoyu Weng3Wei Yan4Jun Song5Tong Ye6Junle Qu7Center for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityCenter for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityCenter for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityCenter for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityCenter for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityCenter for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityDepartment of Bioengineering, CU-MUSC Bioengineering Program, Clemson UniversityCenter for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen UniversityAbstract Multicolor microscopy and super-resolution optical microscopy are two widely used techniques that greatly enhance the ability to distinguish and resolve structures in cellular imaging. These methods have individually transformed cellular imaging by allowing detailed visualization of cellular and subcellular structures, as well as organelle interactions. However, integrating multicolor and super-resolution microscopy into a single method remains challenging due to issues like spectral overlap, crosstalk, photobleaching, phototoxicity, and technical complexity. These challenges arise from the conflicting requirements of using different fluorophores for multicolor labeling and fluorophores with specific properties for super-resolution imaging. We propose a novel multicolor super-resolution imaging method called phasor-based fluorescence spatiotemporal modulation (Phasor-FSTM). This method uses time-resolved detection to acquire spatiotemporal data from encoded photons, employs phasor analysis to simultaneously separate multiple components, and applies fluorescence modulation to create super-resolution images. Phasor-FSTM enables the identification of multiple structural components with greater spatial accuracy on an enhanced laser scanning confocal microscope using a single-wavelength laser. To demonstrate the capabilities of Phasor-FSTM, we performed two-color to four-color super-resolution imaging at a resolution of ~λ/5 and observed the interactions of organelles in live cells during continuous imaging for a duration of over 20 min. Our method stands out for its simplicity and adaptability, seamlessly fitting into existing laser scanning microscopes without requiring multiple laser lines for excitation, which also provides a new avenue for other super-resolution imaging technologies based on different principles to build multi-color imaging systems with the requirement of a lower budget.https://doi.org/10.1038/s41377-024-01711-y |
| spellingShingle | Luwei Wang Yue Chen Jiaqing Guo Xiaoyu Weng Wei Yan Jun Song Tong Ye Junle Qu Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing Light: Science & Applications |
| title | Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing |
| title_full | Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing |
| title_fullStr | Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing |
| title_full_unstemmed | Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing |
| title_short | Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing |
| title_sort | phasor fstm a new paradigm for multicolor super resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing |
| url | https://doi.org/10.1038/s41377-024-01711-y |
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