PDF(Pubmed)
Abstract：
Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels\' fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.
摘要：
荧光标记对生物分析的许多进步做出了巨大贡献，分子生物学,分子成像,和医学诊断。尽管有大量的分子和纳米级荧光团工具箱可供选择，仍然需要更明亮的标签，例如，用于流式细胞术和荧光显微镜，优选具有分子性质。这需要荧光团多聚化的通用概念，这涉及到从其他发色团和可能的猝灭剂在其附近的染料的屏蔽。此外,为了增加荧光显微镜和最终流式细胞术的读出参数的数量，需要控制和调整标签的荧光寿命。搜索明亮的多色或多聚体标签，我们开发了带有用于其生物共轭的官能团的PEG化染料，并与两种示例性选择的在488nm处可激发的荧光团的相应单体染料相比，探索了它们的光谱性质和光稳定性。随后，这些染料与抗CD4和抗CD8免疫球蛋白缀合,以获得适合标记细胞和珠子的荧光缀合物。最后,评估了这些新型标记用于荧光寿命成像和基于寿命测量的目标区分的适用性。基于我们的光谱研究的结果，包括荧光量子产率（QY）和荧光衰减动力学的测量，我们可以证明在这些多聚体标记中不存在明显的染料-染料相互作用和自猝灭。此外，在第一次荧光寿命成像(FLIM)研究中，我们可以展示这种多聚化概念对终生区分和多路复用的未来潜力。
