Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy
Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy
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DOI：
10.1039/c0cp01919d
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发表时间：
2011-01-01
影响因子：
3.3
通讯作者：
Tinnefeld, Philip
中科院分区：
文献类型：
--
作者：
Cordes, Thorben;Maiser, Andreas;Tinnefeld, Philip
现代荧光显微镜应用沿着对所用荧光染料的需求不断增加。在这项工作中，我们比较了抗褪色公式利用最近开发的还原和氧化系统（ROXS）与商业抗褪色剂。为了系统地测试荧光团在生物样品的荧光成像中的性能，我们使用标记有各种常用有机染料（例如Alexa 488、Alexa 594、Alexa 647、Cy3B、ATTO 550和ATTO 647 N）的固定细胞进行光漂白实验。定量评价（i）光稳定性，（ii）亮度，和（iii）在不同的防褪色（AF）中安装的样品中的荧光团的存储稳定性揭示了染料和AF的最佳组合。基于这些结果，我们提供了指导，AF应优先与特定染料一起使用。最后，我们使用单分子光谱学研究了商业AF的抗褪色机制，并揭示了这些经验选择的AF具有与ROXS AF相似的特性。
Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.