Nir Eyal, Michalet Xavier, Hamadani Kambiz M, Laurence Ted A, Neuhauser Daniel, Kovchegov Yevgeniy, Weiss Shimon
Department of Chemistry and Biochemistry, and California NanoScience Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.
J Phys Chem B. 2006 Nov 9;110(44):22103-24. doi: 10.1021/jp063483n.
We describe a simple approach and present a straightforward numerical algorithm to compute the best fit shot-noise limited proximity ratio histogram (PRH) in single-molecule fluorescence resonant energy transfer diffusion experiments. The key ingredient is the use of the experimental burst size distribution, as obtained after burst search through the photon data streams. We show how the use of an alternated laser excitation scheme and a correspondingly optimized burst search algorithm eliminates several potential artifacts affecting the calculation of the best fit shot-noise limited PRH. This algorithm is tested extensively on simulations and simple experimental systems. We find that dsDNA data exhibit a wider PRH than expected from shot noise only and hypothetically account for it by assuming a small Gaussian distribution of distances with an average standard deviation of 1.6 A. Finally, we briefly mention the results of a future publication and illustrate them with a simple two-state model system (DNA hairpin), for which the kinetic transition rates between the open and closed conformations are extracted.
我们描述了一种简单的方法，并提出了一种直接的数值算法，用于在单分子荧光共振能量转移扩散实验中计算最佳拟合散粒噪声限制的接近率直方图（PRH）。关键要素是使用通过光子数据流进行猝发搜索后获得的实验猝发大小分布。我们展示了如何使用交替激光激发方案和相应优化的猝发搜索算法消除影响最佳拟合散粒噪声限制PRH计算的几个潜在伪像。该算法在模拟和简单实验系统上进行了广泛测试。我们发现双链DNA数据表现出比仅由散粒噪声预期的更宽的PRH，并假设通过假设距离的小高斯分布（平均标准差为1.6 Å）来解释这一现象。最后，我们简要提及了未来一篇出版物的结果，并用一个简单的两态模型系统（DNA发夹）进行说明，从中提取了开放和闭合构象之间的动力学转变速率。
