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Published on: June 7, 2019
Fluorescence Lifetime Multiplexing with Fluorogen-Activating FAST Protein Variants and Red-Shifted
Aidar R Gilvanov1,2, Ivan N Myasnyanko1,2, Sergey A Goncharuk1
1Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Abstract:
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 N871b from the arylidene-imidazolone family. We showed that a series of FAST protein mutants exhibit similar steady-state optical properties in complex with N871b fluorogen but have different fluorescence lifetimes. The similar brightness and binding strength of pairs of these FAST protein variants with N871b allows them to be successfully used for multiplexing up to three intracellular structures of living cells simultaneously.
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