MEASURING ANGULAR FLUCTUATIONS WITH POLARIZED FCS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。与核心项目1和几个合作项目一起，我的工作重点是探索使用FCS测量角运动的各种途径。我正在研究这项技术的模拟、分析方法和实际应用。通过仿真，使用SimFCS和我自己的程序，对偏振激发光的使用和偏振发射的测量进行了测试和评估。从最近严格遵循激发和发射矢量的模拟数据集来看，使用非偏振(或圆偏振)激发光似乎可以有效地跟踪单个粒子在FCS点扩散函数中扩散时的旋转。从极化的双通道发射计算极化时间序列允许计算极化自相关曲线，该曲线似乎与平移扩散引起的涨落无关地跟随粒子的角旋转。当我们使用偏振激发光时，这种分离并不完全，正如我们曾经认为的那样。除了模拟，还有旋转缓慢的样品，如dsDNA片段，小单层囊泡中的荧光团，支撑双层中的荧光团运动，以及膜结合蛋白，以评估该技术的实际应用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In conjunction with core project 1 and several collaboration projects, my work has focused on exploring the various avenues for measuring angular motion using FCS. I am working on simulations, analysis methods, and practical application of the technique. Through simulations, using SimFCS and my own programs, I have tested and evaluated the use of polarized excitation light and measuring the polarized emission. From recent simulated data sets in which the excitation and emission vectors are strictly followed, it appears that the use of unpolarized (or circularly polarized) excitation light effectively allows one to follow the rotation of single particles as they diffuse through the FCS point spread function. Calculating the polarization time series from the polarized dual channel emission allows one to calculate the polarization autocorrelation curve that appears to follow the angular particle rotation independent of fluctuations due to translational diffusion. This separation is not complete when one uses polarized excitation light, as we once thought. In addition to simulations, samples with slow rotations, such as dsDNA fragments, fluorophores in small unilamellar vesicles, fluorophore movement in supported bilayers, and membrane-bound proteins are being examined to evaluate the practical application of the technique.