Carborhodol: A New Hybrid Fluorophore Obtained by Combination of Fluorescein and Carbopyronine Dye Cores

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• 作者：Maksim V. Sednev ; Christian A. Wurm ; Vladimir N. Belov ; Stefan W. Hell
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：April 17, 2013
• 年：2013
• 卷：24
• 期：4
• 页码：690-700
• 全文大小：530K
• 年卷期：v.24,no.4(April 17, 2013)
• ISSN：1520-4812

文摘

Asymmetric hybrid fluorophores are built from the structural elements of two (or even more) symmetric dyes and can develop valuable new features which their parents do not possess. A new hybrid carborhodol dye was obtained by the combination of fluorescein and carbopyronine fluorophores. The brightly fluorescent hybrid dye with a linker and reactive group was prepared in 12 steps with overall yield of 1.6%. In aqueous solutions, it has absorption and emission maxima at 586 and 613 nm, respectively. Antibodies labeled with a carborhodol dye possess broad absorption and emission bands so that the effective Stokes shift is increased (compared with small Stokes shifts of the parent dyes) and the fluorescence quantum yield of 39% at a degree of labeling of 5.2. Two samples of secondary antibodies labeled with carborhodol and the benchmark red-emitting rhodamine dye (KK114) were used in two-color imaging experiments with excitation at 514鈥?32 (carborhodol dye) and 633鈥?40 nm (KK114). When emitted light was detected above 650 nm, the novel carborhodol dye provided a lower crosstalk than spectrally similar emitters (e. g., Atto594; crosstalk 40鈥?0% with KK114 under the same conditions). The optical resolution of ca. 80 nm was attained using the new dye in stimulated emission depleted (STED) microscopy. The relatively short fluorescence lifetime in conjugates with antibodies (蟿 = 1.2鈥?.6 ns) suggests the possibility of dual FLIM with numerous dyes having 蟿 values in the range of 3鈥? ns. All of these features make the carborhodol fluorophore a valuable addition to the family of the red-emitting fluorescent dyes.