MEASURING ANGULAR FLUCTUATIONS WITH POLARIZED FCS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。结合核心项目1和几个合作项目，我的工作重点是探索使用FCS测量角运动的各种途径。我正在研究模拟，分析方法和该技术的实际应用。通过模拟，使用SimFCS和我自己的程序，我已经测试和评估了偏振激发光的使用和测量偏振发射。从最近的模拟数据集，其中严格遵循的激发和发射矢量，它出现的非偏振（或圆偏振）激发光的使用有效地允许一个跟随单个粒子的旋转，因为它们扩散通过FCS点扩散函数。从偏振双通道发射计算偏振时间序列允许计算偏振自相关曲线，该曲线似乎遵循独立于由于平移扩散引起的波动的角粒子旋转。正如我们曾经认为的那样，当使用偏振激发光时，这种分离并不完全。除了模拟，样品与缓慢的旋转，如双链DNA片段，荧光团在小的单层囊泡，荧光团运动支持双层，膜结合蛋白质正在检查，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.