Multiparameter microscopy and spectroscopy for single-molecule analytics.
Multiparameter microscopy and spectroscopy for single-molecule analytics.
复制标题
用于单分子分析的多参数显微镜和光谱学。
DOI：
10.1021/ac034976g
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发表时间：
2004
影响因子：
7.4
通讯作者：
U. Wild
中科院分区：
文献类型：
--
作者：
M. Prummer;Beate Sick;A. Renn;U. Wild
从不同的单个荧光报告分子同时监测多个参数的能力有助于解开复杂和相互作用系统的纠缠，并为从基础科学到生物制药技术等领域开辟了新的视角。通过结合环形照明显微镜、时间相关单光子计数和多通道检测，我们能够从一个单独的荧光团确定14个独立的参数。从荧光光子的几个特性，即到达时间、波长和偏振度，推导出了整套参数。利用该方法，可以在纳秒时间尺度上分析强度、极化和光谱动力学，并可以在亚毫秒时间分辨率下监测平均值。据我们所知，这是第一次观察到单分子的纳秒级光谱动力学。根据我们的经验，我们可以确定生物样品中30%以上的照明荧光团和掺杂聚合物薄膜中80%以上的荧光团的所有参数。
The ability to monitor several parameters simultaneously from distinct individual fluorescent reporter molecules facilitates the disentanglement of complex and interacting systems and opens new perspectives in areas from basic science to biopharmaceutical technology. By combining annular illumination microscopy, time-correlated single-photon counting, and multichannel detection, we were able to determine 14 independent parameters from one individual fluorophore. The whole set of parameters was deduced from the few properties of the fluorescence photons, i.e., arrival time, wavelength, and polarization. With this approach, the intensity, the polarization, and the spectral dynamics can be analyzed on a nanosecond time scale and the mean values can be monitored with submillisecond time resolution. Nanosecond spectral dynamics of single molecules has been observed, to the best of our knowledge, for the first time. From our experience, we can determine all parameters for more than 30% of the illuminated fluorophores in biological samples and for more than 80% in doped polymeric films.