Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes.