Development of fluorescence labeling technique for multi-modal live-cell super-resolution microscopy

Abstract

Live-cell super-resolution microscopy is a powerful tool that reveals spatiotemporal dynamics of biomolecules with nano-scale resolution in live specimens. However, the photobleaching of fluorescent molecules labeled to the target molecule is a significant obstacle to continuously obtaining super-resolution images for a long period. In this study, we developed a fluorescent labeling technique called de-quenching of organic dye emission (DeQODE) that enables long-term observation with a super-resolution microscope without being affected by photobleaching. In the DeQODE, a non-fluorescent probe is designed to become fluorescent by sequential binding to tag protein, an antibody against the probe, enabling maintaining the fluorescence signal for a long period. After optimizing the binding-dissociating property between tag protein and the probe, several intracellular organelles and the distribution of proteins labeled with DeQODE were imaged with a nano-scale resolution for tens of minutes by multiple super-resolution microscopies including stimulated emission depletion microscopy (STED), single-molecule localization microscopy (SMLM), and structured illumination microscopy (SIM). Our results show that DeQODE can contribute to research clarifying the nano-scale dynamics of functional molecules by live-cell super-resolution imaging.