Quantum-Yield-Optimized Fluorophores for Site-Specific Labeling and Super-Resolution Imaging

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• 作者：Christian Grunwald ; Katrin Schulze ; Gregory Giannone ; Laurent Cognet ; Brahim Lounis ; Daniel Choquet ; Robert Tamp茅
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：June 1, 2011
• 年：2011
• 卷：133
• 期：21
• 页码：8090-8093
• 全文大小：768K
• 年卷期：v.133,no.21(June 1, 2011)
• ISSN：1520-5126

文摘

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鈥揻luorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA鈥揚PII鈥揻luorophores 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鈥揚PII鈥揻luorophores in super-resolution microscopy.