Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics
Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics
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DOI：
10.1021/jp066082g
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发表时间：
2007-01-18
影响因子：
3.3
通讯作者：
Tinnefeld, Philip
中科院分区：
文献类型：
--
作者：
Ross, Joachim;Buschkamp, Peter;Tinnefeld, Philip
我们已经开发了共聚焦荧光单分子光谱，优化了检测灵敏度的三个光谱不同的通道的研究生物分子之间的相互作用和FRET两个以上的分子。采用可编程声光器件作为分束器和激发滤波器，克服了传统多色分束器的一些局限性，实现了三条激光线的快速交替。这使得可视化的DNA三路交界处的合成在一个单分子的基础上，并解决七个化学计量的亚群，以及量化FRET在竞争的能量转移途径的存在。此外，通过同时监测生物分子复合物内的几个距离来研究相关分子运动的能力被证明。
We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.