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bioRxiv · 10.1101/2023.03.24.534096

Super-Resolved FRET and Co-Tracking in pMINFLUX

Cole, F.·Zaehringer, J.·Bohlen, J.·Schroeder, T.·Steiner, F.·Stefani, F. D.·Tinnefeld, P.
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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 colocalize 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-microtimegating 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/534096v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@11459forg.highwire.dtl.DTLVardef@1f1cee2org.highwire.dtl.DTLVardef@4592e6org.highwire.dtl.DTLVardef@1af3e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Cole, F., Zaehringer, J., Bohlen, J., Schroeder, T., Steiner, F., Stefani, F. D., Tinnefeld, P.. 2023-03-25. Super-Resolved FRET and Co-Tracking in pMINFLUX. https://doi.org/10.1101/2023.03.24.534096

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