Hense Anika, Prunsche Benedikt, Gao Peng, Ishitsuka Yuji, Nienhaus Karin, Nienhaus G Ulrich
Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany.
Institute of Nanotechnology, Karlsruhe Institute of Technology (KIT), 76344 Eggenstein-Leopoldshafen, Germany.
Sci Rep. 2015 Dec 9;5:18006. doi: 10.1038/srep18006.
The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.
绿色荧光蛋白（GFP）远红发射变体的发展对于活细胞、组织和生物体成像至关重要。尽管付出了显著努力，但远红标记蛋白仍需进一步优化以匹配其绿色对应物的性能。在此，我们展示了mGarnet，一种强大的单体标记蛋白，其远红荧光在670nm处达到峰值。由于其95,000 M(-1)cm(-1)的大消光系数，mGarnet可以在其598nm激发带的红边上用640nm光有效激发。大斯托克斯位移使得即使在640nm激发下也能基本上收集到整个荧光发射，抵消了mGarnet较低的荧光量子产率（9.1%），这是远红荧光蛋白的典型特征。我们在受激发射损耗（STED）显微镜中使用640nm激发和780nm损耗波长，证明了其作为活细胞融合标记的优异性能。
