Abstract
Single-molecule fluorescence 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 suffer from the spatial restrictions of FRET, they only visualize one emitter at a time, thus making it difficult 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 fluorescence 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 fluorophores with similar spectral properties but distinct fluorescence 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.
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Data availability
All data are available from the corresponding author on reasonable request.
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Codes used in this study are available from the corresponding author on reasonable request.
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Acknowledgements
P.T. is grateful for support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project-ID 201269156—SFB 1032 Project A13 and funding by the Bavarian Ministry of Science and the Arts through the ONE MUNICH Project ‘Munich Multiscale Biofabrication’. P.T. is indebted to the DFG for funding via grant nos. 470075523 and 459594986, and Germany’s Excellence Strategy (grant no. EXC 2089/1-390776260). We thank P. Luna, P. Xaverl and T. Charly for their support with the measurements and data analysis, and A. Kardinal, H. Lipfert, S. Steger and M. Ehrl for laboratory upkeep.
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F.C., J.Z., T.S., F.S., F.D.S. and P.T developed the concept. F.C., J.Z. and J.B. designed and prepared samples. M.P. designed and prepared antibody samples. P.S. designed and prepared lipid bilayer samples. F.C. and J.Z. performed and analysed measurements. P.T. supervised the project. All authors have written, read and approved the final manuscript.
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Supplementary Figs. 1–16 and Sections 1–14.
Supplementary Video 1
The time evolution of the localizations with the corresponding FRET radii and the subsequent localization of the FRET acceptor.
Supplementary Video 2
The time evolution of the co-localization of two pointer systems by lifetime multiplexing.
Supplementary Video 3
The time evolution of the orientation of the lipid raft measured by the co-localization of two fluorophores by lifetime multiplexing.
Supplementary Data 1
Supplementary Tables 2–9. Sequences of the DNA oligonucleotides used for DNA origami folding.
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Cole, F., Zähringer, J., Bohlen, J. et al. Super-resolved FRET and co-tracking in pMINFLUX. Nat. Photon. (2024). https://doi.org/10.1038/s41566-024-01384-4
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DOI: https://doi.org/10.1038/s41566-024-01384-4