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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| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-04-01
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| Series: | Biosensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6374/15/5/274 |
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| Summary: | 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. |
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| ISSN: | 2079-6374 |