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> 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.
Authors
- Михаил С. Баранов (ORCID: https://orcid.org/0000-0002-9339-7603)
- Aidar R. Gilvanov (ORCID: https://orcid.org/0000-0001-6174-7798)
- Marina V. Goncharuk (ORCID: https://orcid.org/0000-0002-6984-7125)
- Vadim S. Kublitski (ORCID: https://orcid.org/0000-0001-7142-2794)
- Svetlana V. Sidorenko (ORCID: https://orcid.org/0000-0002-4074-0500)
- Ivan N. Myasnyanko (ORCID: https://orcid.org/0000-0002-2168-3555)
- Sergey A. Goncharuk (ORCID: https://orcid.org/0000-0002-0263-6462)
- Eugene G. Maksimov (ORCID: https://orcid.org/0000-0001-6767-1501)
- Yulia A. Bogdanova (ORCID: https://orcid.org/0000-0003-1745-4204)
- Daria V. Bodunova
Institutions
- Lomonosov Moscow State University (RU)
- Pirogov Russian National Research Medical University (RU)
- Institute of Bioorganic Chemistry (RU)
Publication Details
- Journal
- Biosensors
- Published
- 2025-04-29
- DOI
- https://doi.org/10.3390/bios15050274
- Citations
- 2
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 3.27
Funders
- Russian Science Foundation