Authors
Habenstein F, Jensen N, Stumpf D, Chizhik AI, Leutenegger M, Chang J, Fognini A, Qin-Dregely J, Zadeh IE, Kirck LL, Enderlein J, Inamdar K, Hell SW, Jakobs S
Journal
Proceedings of the National Academy of Sciences of the USA
Citation
Proc Natl Acad Sci U S A. 2026 Sep;123(35):e2533451123.
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.
