Bright monomeric fluorescent protein elite-niRFP704 for two-channel near-infrared STED nanoscopy

• 成果类型: Article
• 署名作者: Habenstein, Florian; Jensen, Nickels; Stumpf, Daniel; Chizhik, Alexey I.; Leutenegger, Marcel; Chang, Jin; Fognini, Andreas; Qin-Dregely, Jessie; Zadeh, Iman Esmaeil; Kirck, Laura L.; Enderlein, Jorg; Inamdar, Kaushik; Hell, Stefan W.; Jakobs, Stefan
• 署名单位: University of Gottingen; Delft University of Technology; University of Gottingen; University of Gottingen; Max Planck Society
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2533451123
• 发表日期: 2026-09-01
• 页码: e2533451123
• 关键词: superresolution microscopy; STED; bacteriophytochrome; fluorescent protein; live-cell imaging; mutagenesis; biosensors; light; knot

摘要

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.