Super-Resolved FRET and Co-Tracking in pMINFLUX

Abstract

Single-molecule FRET (smFRET) is widely used to investigate dynamic (bio) molecular interactions taking place over distances of up to 10 nm. With the advent of recent super-resolution methods such as MINFLUX, MINSTED or RASTMIN, the spatiotemporal resolution of these techniques advanced towards the smFRET regime. While these methods do not suffer from the spatial restriction of FRET, they only visualize one emitter at a time, thus rendering fast dynamics of interactions out of reach. Here, we describe two approaches to overcome this limitation in pMINFLUX using its intrinsic fluorescence lifetime information. First, we combined pMINFLUX with smFRET. This enabled us to track a FRET donor fluorophore and simultaneously co-localize its FRET acceptor with nanometer precision. To extend co-localized tracking beyond the FRET range, we developed pMINFLUX lifetime multiplexing, a method to simultaneously track two fluorophores with similar spectral properties but distinct fluorescence lifetimes. We demonstrated its application on both static and dynamic DNA origami systems with a precision better than 2 nm. Within the FRET range, pMINFLUX lifetime multiplexing additionally uses a novel combined phasor-microtime-gating approach. This paves the way for nanometer precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, closing the resolution gap between smFRET and co-tracking.

A Fast Interferometric Beam Shaper for Multi-Emitter 3D MINFLUX

“…It is the key component facilitating 3D and two-color operation for imaging and tracking in the presented MINFLUX system and we believe it will make these features more accessible for future MINFLUX implementations. Furthermore, this technology enabled the first demonstrations of MINFLUX excitation schemes mixing distinct beam shapes for improved precision and concurrent two-emitter MINFLUX tracking in 3D, following the recent introduction of simultaneous two-emitter MINFLUX tracking in 2D 13 . These advancements pave the way for multi-emitter MINFLUX tracking as a method for studying the kinetics and conformational states of flexible protein complexes free from range limitations present in single-molecule FRET 14 and with time resolutions in the sub-millisecond range, surpassing high-speed AFM 15 .…”

“…We developed and characterized a general-purpose four-channel beam-shaping polarization interferometer, specifically designed for enabling 3D imaging and tracking in a two-color excitation MINFLUX system. The implementation of this technology resulted in the first demonstration of concurrent two-emitter MINFLUX tracking in 3D, subsequent to the recent introduction of simultaneous two-emitter MINFLUX tracking in 2D 13 . These advancements pave the way for multi-emitter MINFLUX tracking as a method for studying the kinetics and conformational states of flexible protein complexes with respect to an absolute origin, free from range limitations present in single-molecule FRET 14 and with time resolutions in the sub-millisecond range, surpassing high-speed AFM 15 .…”