Time-resolved fluorescent proteins expand fluorescent microscopy in temporal and spectral domains

Fluorescence microscopy has been widely applied in the life sciences. While intensity as a steady-state signal is widely used, the time-resolved (tr) signal using fluorescence lifetime remains underexplored. Herein, we present a family of time-resolved fluorescent proteins (tr-FPs) with rationally controlled lifetimes. Using a strategy that regulates lifetime without affecting the spectra of FPs, we have developed a series of tr-FPs that cover the visible spectrum and a wide range of lifetimes. The tr-FPs are employed in temporal-spectral resolved microscopy, allowing for the simultaneous imaging of 9 different proteins in live cells and the correlation of multiple activities to cell cycles. Furthermore, tr-FPs enable multiplexing super-resolution microscopy that concurrently visualizes 4 proteins using the lifetime signal and are demonstrated to quantify the stoichiometry of cellular proteins. Our work introduces the concept and development of tr-FPs as a transformative toolset, presenting opportunities to integrate system complexity and quantitative accuracy into biological research.