Stochastic optical reconstruction microscopy (STORM) is a popular method of super-resolution microscopy due to moderate requirements on the optical setup and high achievable resolution. However, since its inception more than 15 years ago, protocols have barely evolved, and despite some recent progress, multicolor imaging can still be complex without the right equipment. To achieve better multicolor images, we decided to optimize the buffer composition to improve the blinking of the most popular red dye CF-568 while maintaining good performance for far-red fluorophores such as Alexa-647 using four optimization parameters, namely, the concentration of three chemicals and the pH. We developed a simple, inexpensive, and stable buffer that can be stored for several weeks and frozen for longer-term storage that allows high-quality three-color STORM imaging.

STORM microscopy is one of the most popular method of super-resolution microscopy, due to moderate requirements on the optical setup, and high achievable resolution. However, since its inception more than 15 years ago, protocols have barely evolved, and despite some recent progress, multicolor imaging can still be complex without the right equipment. We decided to optimize the buffer composition to improve the blinking of the most popular red dye CF-568 while maintaining good performance for far-red fluorophores such as Alexa-647 using the concentration of three chemicals and the pH as 4 optimization parameters. We developed a simple, cheap and stable buffer, that can be stored several weeks and frozen for longer term storage that allow high quality 3-color STORM imaging.