Multi-color, quantitative single-molecule localization microscopy (qSMLM) based on kinetic analysis of fluorophore blinking cycles

我们将建立多色单分子超分辨成像与纳米级定量读出的混合技术，称为qSMLM。这项工作将利用SMLM实验中产生的独特类型的原始数据，即单个发射器的坐标和探测事件的时间。在大多数情况下，这些数据用于重建具有亚衍射空间分辨率的图像。然而，它也包含有价值的光动力学信息：光活化/光切换荧光蛋白和荧光团表现出闪烁的光物理现象（荧光间歇性），这遵循明确的动力学定律，并允许确定多分子复合物中存在多少荧光团（Fricke等人，2015）。单个荧光团闪烁的时间过程可以直接从SMLM实验的原始数据中得到。我们的目标是通过同时生成超分辨率图像，并通过分析眨眼动力学来确定蛋白质化学计量，建立多色定量SMLM （qSMLM）。我们将研究荧光蛋白和有机荧光团结合各种标记技术进行蛋白质标记。为了校准和验证目的，我们将利用定义良好的膜蛋白和同源寡聚物标准（Finan等人，2015）以及DNA折纸（Schmied等人，2012）。我们的方法将允许在分子水平上提取定量信息，这对于任何应用单分子超分辨率显微镜的人来说都是非常有用的扩展。这对于研究完整细胞质膜内的同源和异寡聚信号蛋白复合物具有重要的分子分辨率。

We will establish the hybrid technology of multi-color single-molecule super-resolution imaging with quantitative read-out at the nanoscale, termed qSMLM. This effort will exploit the unique type of raw data generated in an SMLM experiment, i.e. the coordinates of single emitters and the time of the detection event. In most cases, this data is used to reconstruct an image with subdiffraction spatial resolution. However, it also contains valuable photokinetic information: photoactivatable/photoswitchable fluorescent proteins and fluorophores exhibit the photophysical phenomenon of blinking (a fluorescence intermittency), which follows well-defined kinetic laws and allows to determine how many fluorophores are present in a multi-molecular complex (Fricke et al., 2015). The time course of blinking of a single fluorophore is directly available from the raw data of an SMLM experiment. We aim to establish multi-color quantitative SMLM (qSMLM) by simultaneously generating super-resolution images and determining protein stoichiometries by analyzing the kinetics of blinking. We will investigate fluorescent proteins and organic fluorophores in combination with a variety of tagging technologies for protein labeling. For calibration and validation purposes, we will exploit well-defined standards of membrane proteins and homo-oligomers (Finan et al., 2015) as well as DNA origami (Schmied et al., 2012). Our approach will allow extracting quantitative information at the molecular level, representing an extremely useful extension for anyone applying single-molecule super-resolution microscopy. It will in particular be useful to study homo- and heterooligomeric signaling protein complexes in the plasma membrane of an intact cell with molecular resolution.