Abstract
Super-resolution microscopy has greatly enhanced our understanding of nano-scale molecular complexes, such as in the central nervous and immune systems. However, rapid photobleaching of fluorescent dye has hampered live-cell time-lapse super-resolution imaging for long-term nano-scale observations. In this study, we developed the "De-Quenching of Organic Dye Emission" (DeQODE) system to analyze nano-scale molecular distribution dynamics precisely. DeQODE system is a renewable fluorescence labeling technique designed to overcome photobleaching. It comprises an organic probe with a quencher moiety and a fluorescence dye moiety (QODE probe), and an intracellularly expressible single-chain antibody against the quencher (DeQODE tag). The reversible binding between the QODE probe and the DeQODE tag allows fluorogenic molecular labeling to maintain fluorescence signals during imaging experiments.
Using the DeQODE system, we achieved time-lapse super-resolution imaging by stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and super-resolution radial fluctuations (SRRF) method in cultured cells which express DeQODE tag-fused proteins. Additionally, at low QODE probe concentrations, we successfully performed single-particle tracking of labeled proteins sequentially labeled with fluorescence, obtaining trajectory data for more than 10 minutes. Our results suggest that the DeQODE system is promising for analyzing cellular functions, focusing on the nano-scale molecular distribution.
