PcSOFI as a smart label-based superresolution microscopy technique.

Stochastic optical fluctuation imaging (SOFI) is a superresolution imaging technique that uses the flickering of fluorescent labels to generate a microscopic image with a resolution better than what the diffraction limit allows. Its adaptation towards fluorescent protein-labeled samples (called photoconversion SOFI or pcSOFI) allows for a straightforward and easily accessible way of generating superresolution images. In this protocol, we will discuss how so-called "smart labels," and specifically the reversibly switchable fluorescent proteins, have opened doors towards superresolution imaging in general and we provide a protocol on how to perform pcSOFI on HeLa cells expressing human β-actin labeled with the reversibly photoswitchable fluorescent protein Dronpa.