MEASURING ANGULAR FLUCTUATIONS WITH POLARIZED FCS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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。我正在研究该技术的模拟、分析方法和实际应用。通过模拟，使用SimFCS和自己编写的程序，对偏振激发光的使用进行了测试和评估，并测量了偏振发射。从最近严格遵循激发和发射矢量的模拟数据集来看，似乎使用非偏振（或圆偏振）激发光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。从极化双通道发射计算极化时间序列，可以计算出极化自相关曲线，该曲线与粒子角旋转无关，不受平移扩散引起的波动的影响。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。除了模拟之外，正在检查具有缓慢旋转的样品，例如dsDNA片段、小单层囊泡中的荧光团、支持双层中的荧光团运动和膜结合蛋白，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.