Super-resolved FRET and co-tracking in pMINFLUX

Abstract

Single-molecule fuorescence resonance energy transfer (smFRET) is widely used to investigate dynamic (bio)molecular interactions occurring over distances of up to 10 nm. Recent advances in super-resolution methods have brought their spatiotemporal resolution closer towards the smFRET regime. Although these methods do not sufer from the spatial restrictions of FRET, they only visualize one emitter at a time, thus making it difcult to capture fast dynamics of the interactions. Here we describe two approaches to overcome this limitation in pulsed-interleaved MINFLUX (pMINFLUX) microscopy by using its intrinsic fuorescence lifetime information. First we combine pMINFLUX with smFRET, which enables tracking a FRET donor with nanometre precision while simultaneously determining its distance to a FRET acceptor, yielding the acceptor position by multilateration. Second, we developed pMINFLUX lifetime multiplexing—a method that simultaneously tracks two fuorophores with similar spectral properties but distinct fuorescence lifetimes—to extend co-localized tracking beyond the FRET range. We demonstrate applications on DNA origami systems as well as by imaging the paratopes of an antibody with precision better than 2 nm, paving the way for nanometre precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, and closing the resolution gap between smFRET and co-tracking.