Optical Switchable Spironaphthoxazine (NISO)-derived Probes for Optical Lock-in Detection (OLID) Imaging Microscopy and OLID-FRET

An optical switch probe based on spironaphthoxazine (NISO) harboring a O6-benzylguanine (BG) functionality was synthesized and characterized for applications in optical lock-in detection (OLID) Image microscopy. NISO undergoes rapid and reversible, high-fidelity transitions between a colorless spiro (SP) state and a blue colored MC-state that serves as an acceptor probe in Forster resonance energy transfer (FRET) with GFP, YFP and other green or red emitting donor probes. The transition from SP to MC is brought about with high quantum yield by exciting SP with 365 nm or 720 nm (2-photon) while the MC to SP transition is rapidly affected upon excitation of MC with 543∼632 nm light. Thus a defined waveform of optical or opto-thermal manipulations of the NISO switch provides a simple means to modulate the intensity of a donor probe via FRET. NISO was linked to BG via a polyethylene glycol (PEG) linker and this substrate was shown to efficiently label O6-benzylguanine-DNA alkyltransferase (AGT) and AGT-fusion proteins. The optical switching properties BG-PEG-NISO coupled to AGT in fusion proteins with GFP (GFP-AGT/PEG-NISO) and mCherry-(mCherry-AGT/PEG-NISO) were studied. In vitro OLID-FRET imaging studies showed the suitability of NISO as an acceptor probe for GFP and mcherry. The R0 for FRET between GFP and MC-NISO is 4.7 nm and GFP-AGT fusion protein covalently labeled with BG-PEG-NISO exhibited a 55% decrease in GFP fluorescence intensity upon conversion of SP to MC. Other examples of in vitro and in vivo OLID-FRET using GFP-AGT and mcherry-AGT labeled with NISO-BG will be presented.