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−1cm−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.
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机译：绿色荧光蛋白（GFP）的远红外发射变体的进展对于成像活细胞，组织和生物至关重要。尽管作出了显着努力，但远红色标记蛋白仍需要进一步优化以匹配其绿色对应蛋白的性能。在这里，我们介绍了mGarnet，这是一种坚固的单体标记蛋白，在670nm处具有远红色荧光峰。由于其消光系数高达95,000 M −1 cm -1 ，mGarnet可以在其598 nm激发带的红色边缘上通过640 nm光有效地激发。较大的斯托克斯频移即使在640 nm激发下也能基本上收集所有荧光发射，从而抵消了mGarnet的较低荧光量子产率（9.1％），这是远红FP的典型特征。我们展示了在640 nm激发和780 nm耗尽波长下在STED显微镜中作为活细胞融合标记物的优异性能。
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