Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen
Fluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen <b>N871b</b&g...
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| Format: | Article |
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MDPI AG
2025-04-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/15/5/274 |
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| author | Aidar R. Gilvanov Ivan N. Myasnyanko Sergey A. Goncharuk Marina V. Goncharuk Vadim S. Kublitski Daria V. Bodunova Svetlana V. Sidorenko Eugene G. Maksimov Mikhail S. Baranov Yulia A. Bogdanova |
| author_facet | Aidar R. Gilvanov Ivan N. Myasnyanko Sergey A. Goncharuk Marina V. Goncharuk Vadim S. Kublitski Daria V. Bodunova Svetlana V. Sidorenko Eugene G. Maksimov Mikhail S. Baranov Yulia A. Bogdanova |
| author_sort | Aidar R. Gilvanov |
| collection | DOAJ |
| description | Fluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen <b>N871b</b> from the arylidene–imidazolone family. We showed that a series of FAST protein mutants exhibit similar steady-state optical properties in complex with <b>N871b</b> fluorogen but have different fluorescence lifetimes. The similar brightness and binding strength of pairs of these FAST protein variants with <b>N871b</b> allows them to be successfully used for multiplexing up to three intracellular structures of living cells simultaneously. |
| format | Article |
| id | doaj-art-b85fb7583cb346b3a1ed3186a8ea3a82 |
| institution | OA Journals |
| issn | 2079-6374 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj-art-b85fb7583cb346b3a1ed3186a8ea3a822025-08-20T02:33:43ZengMDPI AGBiosensors2079-63742025-04-0115527410.3390/bios15050274Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as FluorogenAidar R. Gilvanov0Ivan N. Myasnyanko1Sergey A. Goncharuk2Marina V. Goncharuk3Vadim S. Kublitski4Daria V. Bodunova5Svetlana V. Sidorenko6Eugene G. Maksimov7Mikhail S. Baranov8Yulia A. Bogdanova9Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaDepartment of Biology, Lomonosov Moscow State University, Leninskye Gory, Build. 12, Moscow 119234, RussiaDepartment of Biology, Lomonosov Moscow State University, Leninskye Gory, Build. 12, Moscow 119234, RussiaDepartment of Biology, Lomonosov Moscow State University, Leninskye Gory, Build. 12, Moscow 119234, RussiaInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, RussiaFluorescence-lifetime imaging microscopy (FLIM) is a powerful technique for highly multiplexed imaging in live cells. In this work, we present a genetically encoded FLIM multiplexing platform based on a combination of fluorogen-activating protein FAST and red-shifted fluorogen <b>N871b</b> from the arylidene–imidazolone family. We showed that a series of FAST protein mutants exhibit similar steady-state optical properties in complex with <b>N871b</b> fluorogen but have different fluorescence lifetimes. The similar brightness and binding strength of pairs of these FAST protein variants with <b>N871b</b> allows them to be successfully used for multiplexing up to three intracellular structures of living cells simultaneously.https://www.mdpi.com/2079-6374/15/5/274arylidene–imidazoloneFLIMfluorogen-activating protein |
| spellingShingle | Aidar R. Gilvanov Ivan N. Myasnyanko Sergey A. Goncharuk Marina V. Goncharuk Vadim S. Kublitski Daria V. Bodunova Svetlana V. Sidorenko Eugene G. Maksimov Mikhail S. Baranov Yulia A. Bogdanova Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen Biosensors arylidene–imidazolone FLIM fluorogen-activating protein |
| title | Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen |
| title_full | Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen |
| title_fullStr | Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen |
| title_full_unstemmed | Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen |
| title_short | Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted Arylidene–Imidazolone Derivative as Fluorogen |
| title_sort | fluorescence lifetime multiplexing with fluorogen activating fast protein variants and red shifted arylidene imidazolone derivative as fluorogen |
| topic | arylidene–imidazolone FLIM fluorogen-activating protein |
| url | https://www.mdpi.com/2079-6374/15/5/274 |
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