Far-Red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy
Far-Red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy
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DOI：
10.1016/j.bpj.2010.04.025
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发表时间：
2010-07-21
影响因子：
3.4
通讯作者：
Verkhusha, Vladislav V.
中科院分区：
文献类型：
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作者：
Morozova, Kateryna S.;Piatkevich, Kiryl D.;Verkhusha, Vladislav V.
深部组织和整个动物成像以及红色波长范围内的多色标记需要远红荧光蛋白，以及流式细胞术和荧光显微镜中可使用标准红色激光激发的探针。基于受激发射损耗方法的快速发展的超分辨率显微镜还需要基因编码的单体探针在分子水平上标记细胞内蛋白质。基于单体 mKate 变体，我们开发了一种远红 TagRFP657 蛋白，其激发/发射最大值为 611/657 nm。与现有单体远红蛋白相比，TagRFP657 具有多种优势，包括更高的光稳定性、更好的 pH 稳定性、更低的残留绿色荧光以及更高的红色激光激发效率。与其他远红蛋白相比，红移激发和发射光谱允许在流式细胞术和荧光显微镜中同时使用 TagRFP657 与橙色或近红色荧光蛋白。 TagRFP657 被证明是一种有效的蛋白质标签，可使用市售的受激发射损耗显微镜进行超分辨率荧光成像。
Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or near-red fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.