Bright monomeric fluorescent protein elite-niRFP704 for two-channel near-infrared STED nanoscopy
Florian Habenstein (Max Planck Institute for Multidisciplinary Sciences)
Nickels Jensen (Max Planck Institute for Multidisciplinary Sciences)
Daniel Stumpf (Max Planck Institute for Multidisciplinary Sciences)
Alexey I. Chizhik (Georg-August-University)
Marcel Leutenegger (Max Planck Institute for Multidisciplinary Sciences)
Jin Chang (TU Delft - Applied Sciences)
Andreas Fognini (Single Quantum)
Jessie Qin-Dregely (Single Quantum)
Iman Esmaeil Zadeh (TU Delft - Applied Sciences)
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Abstract
The near-infrared (NIR) spectral region is attractive for live-cell imaging, due to low autofluorescence and reduced phototoxicity. Some phytochrome-derived fluorescent proteins absorb and emit fluorescence in the NIR, but have short fluorescence lifetimes and relatively low quantum yields, requiring higher laser powers thus limiting their usefulness for live-cell superresolution microscopy. Using the bacterial phytochrome miRFP703 as a template, we screened for variants with longer fluorescence lifetimes, because the quantum yield and fluorescence lifetime are linked. We identified the bright monomeric fluorescent protein elite-niRFP704, which has a longer lifetime (1.12 ns) and a correspondingly higher quantum yield (0.21) than its template, absorbing and emitting completely in the NIR spectral region. elite-niRFP704 was used to tag proteins in living cells and facilitated extended stimulated emission depletion (STED) microscopy on cell lines stably expressing a fusion protein. Finally, elite-niRFP704 and miRFP703 could be separated based on their significantly different lifetimes, enabling two-channel NIR STED microscopy of living mammalian cells.
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