Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden.
Nat Commun. 2020 Jan 13;11(1):239. doi: 10.1038/s41467-019-13897-6.
Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~10 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.
明亮的单体近红外（NIR）荧光蛋白（FPs）作为多色显微镜和体内成像的蛋白质标签需求量很大。在这里，我们应用合理的设计来工程化一套完整的单体 NIR FPs，它们是最亮的遗传编码 NIR 探针。我们证明，增强型 miRFP 系列 NIR FPs 结合了哺乳动物细胞中的高有效亮度和单体状态，在具有衍射极限的受激发射损耗（STED）显微镜的纳米级成像和小鼠中的厘米级成像中表现良好。在 STED 中，我们在活细胞中实现了约 40nm 的分辨率。在活体小鼠中，我们在深部组织中检测到约 10 个荧光细胞。使用光谱上不同的单体 NIR FP 变体，我们进行双色活细胞 STED 显微镜和双色体内成像。下一代 miRFP 的发射峰从 670nm 到 720nm，应成为多功能 NIR 探针，可在不同模式下在不同空间尺度上进行多路复用成像。
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