A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.
A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.
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DOI：
10.1038/nbt.3550
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发表时间：
2016-07
影响因子：
46.9
通讯作者：
Lin MZ
中科院分区：
文献类型：
--
作者：
Chu J;Oh Y;Sens A;Ataie N;Dana H;Macklin JJ;Laviv T;Welf ES;Dean KM;Zhang F;Kim BB;Tang CT;Hu M;Baird MA;Davidson MW;Kay MA;Fiolka R;Yasuda R;Kim DS;Ng HL;Lin MZ
橙红色荧光蛋白 (FP) 广泛应用于基于 GFP 探针的多重落射荧光显微镜的生物医学研究，但它们不同的激发要求使得采用新的先进显微镜方法进行多重分析变得困难。另外，由于橙红光的相对组织透射率，橙红 FP 可用于哺乳动物的深层组织成像，但它们对照明的依赖性限制了它们作为深层组织中的报告基因的灵敏度。在这里，我们描述了 CyOFP1，一种明亮的工程橙红色 FP，可被青色光激发。我们证明，CyOFP1 能够在单光子和双光子显微镜中使用基于 GFP 的探针进行单激发多重成像，包括光片系统中的延时成像。 CyOFP1 还可以作为高催化蓝光荧光素酶 NanoLuc 共振能量转移的有效受体。 CyOFP1 和 NanoLuc 的优化融合体称为 Antares，在体内充当高度灵敏的生物发光报告基因，从深层组织产生比萤火虫荧光素酶和其他生物发光蛋白更亮的信号。
Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.