Molecule counts in complex oligomers with single-molecule localization microscopy
Molecule counts in complex oligomers with single-molecule localization microscopy
复制标题
DOI：
10.1088/1361-6463/ab3b65
复制
发表时间：
2019-11-20
影响因子：
3.4
通讯作者：
Malkusch, Sebastian
中科院分区：
文献类型：
--
作者：
Baldering, Tim N.;Bullerjahn, Jakob T.;Malkusch, Sebastian
单分子定位显微镜可以解析细胞中的纳米级蛋白质簇，此外还可以从这些簇中提取蛋白质拷贝数。这种分子计数的一个强大的方法是使用随机模型函数的荧光团闪烁的分析。在这里，我们开发了一个理论模型的PALM数据，占检测效率的定量分析。通过这一点，我们能够可靠地提取不同低聚物的群体和复杂的混合物。我们证明了这种方法分析模拟的PALM数据的光活化荧光蛋白。我们生成的低聚物和低聚物的混合物的闪烁数据的模拟，并显示出强大的低聚物识别。此外，我们证明了这种方法的实验PALM数据。
Single-molecule localization microscopy resolves nano-scale protein clusters in cells, and in addition can extract protein copy numbers from within these clusters. A powerful approach for such molecular counting is the analysis of fluorophore blinking using stochastic model functions. Here, we develop a theoretical model for quantitative analysis of PALM data that accounts for the detection efficiency. By this, we are able to extract populations of different oligomers reliably and in complex mixtures. We demonstrate this approach analyzing simulated PALM data of a photoactivatable fluorescent protein. We generate simulations of blinking data of oligomers and of mixtures of oligomers, and show robust oligomer identification. In addition, we demonstrate this approach for experimental PALM data.