title: Quantum-yield-optimized fluorophores for site-specific labeling and super-resolution imaging.
abstract: 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.
authors.affiliation: Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M., Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Grunwald
authors.forename: Katrin
authors.initials: K
authors.lastname: Schulze
authors.forename: Gregory
authors.initials: G
authors.lastname: Giannone
authors.forename: Laurent
authors.initials: L
authors.lastname: Cognet
authors.forename: Brahim
authors.initials: B
authors.lastname: Lounis
authors.forename: Daniel
authors.initials: D
authors.lastname: Choquet
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006624:Hippocampus / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000737:chemistry; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D018091:Receptors, AMPA