Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

3-POS Board 1 INVESTIGATING LOCAL PAINT BY SINGLE-MOLECULE ENERGY TRANSFER Samrat Basak 1 ; Christoph Schmidt 1 ; Philip Tinnefeld 1 ; 1 Ludwig-Maximilians-Universität München, München, Germany Single-molecule localization microscopy (SMLM) 1 provides detailed structural information overcoming the diffraction limit. Local PAINT 2 is an emerging variant that accelerates nanoscale imaging of clusters by tethering the imaging strand on one site to the cluster and using the other side of the imager to probe transient binding sites in the environment by DNA PAINT. . In Local PAINT (LPAINT) 2 ,imaging depends on the tethered probe to continuously alternate between an unbound state and binding to nearby docking sites. The kinetics of this process determine the sampling rate and ultimately the time required to reconstruct a structure, yet the unbound state has so far remained experimentally inaccessible. Long unbound times could also yield erroneous localizations. Here, we use single-molecule FRET (smFRET) 3 to resolve the full LPAINT probing cycle. This allows us to determine the lifetime of the unbound state, the occupancy of individual docking sites and the transition matrix between them. The measurements therefore reveal how efficiently a tethered probe searches its local environment and how this search translates into localization events. We apply this approach to advance the LPAINT variant Nunchuck LPAINT (NuLPAINT), which combines rigid and flexible DNA elements to probe sites separated by distances from a few to tens of nanometers. In recent work we combined FRET with pMINFLUX 4 illustratings how molecular-state information may ultimately be linked to nanometer-resolved position measurements. Access to the unbound state provides a basis for understanding and optimizing LPAINT, connecting single-molecule kinetics with localization microscopy and revealing how probe dynamics determine the speed and efficiency of nanoscale structural mapping.1. Lelek, M. et al. Single-molecule localization microscopy. Nat. Rev. Methods Primer 1, 39 (2021).2. Zähringer, J. et al. Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super resolution microscopy. Light Sci. Appl. 12, 70 (2023).3. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl. Acad. Sci. 93, 6264–6268 (1996). 4. Masullo, L. A. et al. Pulsed Interleaved MINFLUX. Nano Lett. 21, 840–846 (2021).

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