TIME-RESOLVED LUMINESCENCE RESONANCE ENERGY TRANSFER IMAGING OF PROTEIN-PROTEIN INTERACTIONS IN LIVING CELLS

Lanthanide-based or luminescence resonance energy transfer (LRET) microscopy can be used to sensitively image interactions between reporter-labeled proteins in living mammalian cells. With LRET, luminescent lanthanide complexes are used as donors, conventional fluorophores are used as acceptors, and donor-sensitized acceptor emission occurs at time scales that reflect the long (similar to ms) lanthanide emission lifetime. These long-lived signals can be separated from short-lifetime (similar to ns) sample autofluorescence and directly excited acceptor fluorescence by using pulsed light to excite the specimen and by implementing a short delay (>100 ns) before detection, thereby increasing measurement sensitivity. As practical implementation of time-resolved LRET microscopy requires several potentially unfamiliar experimental techniques, we explicitly describe herein methods to label proteins in living mammalian cells with luminescent terbium complexes, image interactions between terbium-labeled proteins and green fluorescent protein fusions, and quantitatively analyze LRET images.