Mobility Analysis of Super-Resolved Proteins on Optically Stretched DNA: Comparing Imaging Techniques and Parameters
Mobility Analysis of Super-Resolved Proteins on Optically Stretched DNA: Comparing Imaging Techniques and Parameters
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DOI：
10.1002/cphc.201300813
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发表时间：
2014-03-17
期刊：
影响因子：
2.9
通讯作者：
Peterman, Erwin J. G.
中科院分区：
文献类型：
--
作者：
Heller, Iddo;Sitters, Gerrit;Peterman, Erwin J. G.
荧光显微镜结合光镊非常适合研究DNA上的蛋白质迁移率。在这里，我们评估的优点和缺点的超分辨率和常规成像技术的分析一维（1D）的蛋白质扩散通常观察到的DNA结合蛋白。特别是，我们证明了可视化的DNA结合蛋白质使用宽场，共聚焦，和受激发射损耗（STED）显微镜。我们审查这些技术的适用性高蛋白质密度的条件下，并量化其性能的空间和时间分辨率。追踪DNA上的蛋白质迫使人们通过改变成像方式、激发强度和采集速率，在定位精度与定位数量和速率之间做出选择。使用模拟的扩散数据，我们量化这些成像条件的影响，一维扩散分析的准确性。此外，我们考虑的情况下，扩散局限于当地的路障，一个特别相关的蛋白质结合到DNA的情况。这些结果共同提供了指导方针，可以帮助明智地优化DNA和其他一维系统上蛋白质迁移率分析所需的实验条件。
Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super-resolution and conventional imaging techniques for the analysis of one-dimensional (1D) protein diffusion as commonly observed for DNA-binding proteins. In particular, we demonstrate the visualization of DNA-bound proteins using wide-field, confocal, and stimulated emission depletion (STED) microscopy. We review the suitability of these techniques to conditions of high protein density, and quantify their performance in terms of spatial and temporal resolution. Tracking proteins on DNA forces one to make a choice between localization precision on the one hand, and the number and rate of localizations on the other, by altering imaging modality, excitation intensity, and acquisition rate. Using simulated diffusion data, we quantify the effect of these imaging conditions on the accuracy of 1D diffusion analysis. In addition, we consider the case of diffusion confined between local roadblocks, a case particularly relevant for proteins bound to DNA. Together these results provide guidelines that can assist in judiciously optimizing the experimental conditions required for the analysis of protein mobility on DNA and other 1D systems.