Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy

Abstract

The recently proposed fluorescence emission difference (FED) microscopy has been demonstrated to be capable of breaking the diffraction barrier, which restricts the spatial resolution of far-field fluorescence microscopy. In this paper, we report a novel high-speed FED system that can realize real-time super-resolution imaging. By replacing the conventional nanopositioning stage with a galvo mirror, the temporal resolution of FED is improved to nearly one frame per second, which is 100 times faster than that of best existing FED, while the system maintains a super-high spatial resolution of 150 nm, which is far beyond the diffraction barrier. Therefore, the high-speed FED is suitable for large-area observations while avoiding photobleaching. Detailed theoretical analysis, simulations, and experimental real-time resolution tests on 100 nm nanoparticles and biological cells are reported.