Quantum-Yield-Optimized Fluorophores for Site-Specific Labeling and Super-Resolution Imaging
Quantum-Yield-Optimized Fluorophores for Site-Specific Labeling and Super-Resolution Imaging
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DOI：
10.1021/ja200967z
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发表时间：
2011-06-01
影响因子：
15
通讯作者：
Tampe, Robert
中科院分区：
文献类型：
--
作者：
Grunwald, Christian;Schulze, Katrin;Tampe, Robert
单分子应用、饱和模式激发显微镜和受激发射损耗 (STED) 显微镜需要明亮且高度稳定的荧光染料。在这里，我们描述了量子产率优化荧光团的合成，用于蛋白质或大分子复合物的可逆、位点特异性标记。我们使用聚脯氨酸-II (PPII) 螺旋作为具有不同长度的足够刚性的间隔物，以改善一组不同 trisNTA 荧光团的荧光信号。稳态和荧光寿命分析证明了量子产率的提高。作为原理证明，我们在活体神经元突触的体内蛋白质标记和超分辨率成像方面表征了 trisNTA-PPII 荧光团。通过共聚焦和 STED 显微镜对空间受限的突触间隙中 His 标记的 AMPA 受体 (GluA1) 的分布进行成像。荧光强度分布的比较揭示了 STED 显微镜的卓越分辨率。这些结果凸显了生物相容性，特别是小型且光稳定的 trisNTA-PPII 荧光团在超分辨率显微镜中的优势。
Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy.