Protocol for generation and regeneration of PEG-passivated slides for single-molecule measurements

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). [Display omitted] •Regeneration of PEG-passivated slide is simple, quick, and cost effective•Multiple experiments can be performed in a single channel•Different strategies are implemented for different level of regeneration•Regeneration leads to highly reproducible results 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.