Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Physics Frontier Center, Center for the Physics of Living Cells, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
STAR Protoc. 2022 Feb 3;3(1):101152. doi: 10.1016/j.xpro.2022.101152. eCollection 2022 Mar 18.
Single-molecule fluorescence detection by total internal reflection microscope requires surface passivation by polyethylene glycol (PEG) coating, which is labor intensive and is only good for one or two experiments. Here, we present an efficient and reliable protocol for generating and regenerating the PEG surface for multiple rounds of experiments (∼5-10 times) in the same channel. This protocol is very simple, robust, rapid, and versatile; i.e., multiple strategies can be implemented to regenerate different layers of surface. The regeneration strategy saves time, improves the cost effectiveness, and enhances the efficiency of single-molecule experiments. For complete details on the use and execution of this profile, please refer to Paul et al. (2021a).
通过全内反射显微镜进行单分子荧光检测需要通过聚乙二醇（PEG）涂层进行表面钝化，这是一项劳动密集型工作，并且只适用于一到两个实验。在这里，我们提出了一种高效可靠的方案，可在同一通道中进行多次实验（约 5-10 次）来生成和再生 PEG 表面。该方案非常简单、稳健、快速且多功能；即，可以实施多种策略来再生不同的表面层。这种再生策略节省了时间，提高了成本效益，并提高了单分子实验的效率。如需详细了解此方案的使用和执行，请参考 Paul 等人（2021a）的研究。
