Nino Daniel, Rafiei Nafiseh, Wang Yong, Zilman Anton, Milstein Joshua N
Department of Physics, University of Toronto, Toronto, Ontario, Canada; Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada.
Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Biophys J. 2017 May 9;112(9):1777-1785. doi: 10.1016/j.bpj.2017.03.020.
Superresolved localization microscopy has the potential to serve as an accurate, single-cell technique for counting the abundance of intracellular molecules. However, the stochastic blinking of single fluorophores can introduce large uncertainties into the final count. Here we provide a theoretical foundation for applying superresolved localization microscopy to the problem of molecular counting based on the distribution of blinking events from a single fluorophore. We also show that by redundantly tagging single molecules with multiple, blinking fluorophores, the accuracy of the technique can be enhanced by harnessing the central limit theorem. The coefficient of variation then, for the number of molecules M estimated from a given number of blinks B, scales like ∼1/N, where N is the mean number of labels on a target. As an example, we apply our theory to the challenging problem of quantifying the cell-to-cell variability of plasmid copy number in bacteria.
超分辨定位显微镜有潜力成为一种用于计数细胞内分子丰度的精确单细胞技术。然而，单个荧光团的随机闪烁会给最终计数带来很大的不确定性。在此，我们基于单个荧光团闪烁事件的分布，为将超分辨定位显微镜应用于分子计数问题提供了理论基础。我们还表明，通过用多个闪烁荧光团对单个分子进行冗余标记，可以利用中心极限定理提高该技术的准确性。对于从给定数量的闪烁B估计出的分子数M，变异系数的缩放比例约为1/N，其中N是目标上标记的平均数量。作为一个例子，我们将我们的理论应用于量化细菌中质粒拷贝数的细胞间变异性这一具有挑战性的问题。
