RsCherryRev and NISO Red-shifted Optical Switch Probes for Optical Lock-in Detection (OLID) Imaging and 2-colour OLID-FRET

Abstract

Optical lock-in detection (OLID) microscopy using synthetic or genetically encoded optical switches is was developed by our group to generate high-contrast images of the distributions and interactions of proteins in the presence of high and time-varying background signals such as those found in living cells in culture and in live tissue. OLID requires an optical switch probe whose fluorescence intensity can be modulated through deterministic optical control of its fluorescent and non-fluorescent states. Our initial studies focused on Dronpa, while in this study we show how the genetically-encoded optical switch rsCherryRev can be used to extend the wavelength region of OLID to the red (>550 nm) and for 2-colour OLID imaging in combination with Dronpa.Optical lock in detection (OLID) of Foerster resonance energy transfer, OLID-(FRET) using optical switches as acceptor probes can overcome several limitations of FRET imaging of protein interactions in living cells, including detecting low levels of protein complexes that result from endogenous unlabeled proteins, and non-stoichiometric formation of protein complexes between donor and acceptor probes. We have previously used NitroBIPS as an optically switchable acceptor probe for GFP, and in this study we show how the synthetic, red-shifted optical switch spironaphthoxazine (NISO) serves as a switchable acceptor for both GFP and mCherry in OLID-FRET. These new probes are used for 2-colour OLID-FRET of protein interactions in living cells.