Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

52-POS Board 26 MAPPING STRUCTURALLY VARIABLE DNA CONSTRUCTS WITH FRET NANOSCOPY AND OPTICAL PYTHAGORAS Noah H Salama 1 ; Nicolaas T M van der Voort 1,6 ; Jan-Hendrik Budde 1 ; Michelle P Rademacher 1,2 ; Suren Felekyan 1 ; Christian A Hanke 1 ; Ralf Kühnemuth 1 ; Julian Folz 1,7 ; Markus Köhler 3 ; Andreas Schönle 3 ; Alexander Henrichs 1 ; Julian Sindram 4 ; Hilke E K Werner 1 ; Marius Otten 4 ; Chiara S Pulver 1 ; Sonja L Osten 1 ; Pauline Rademacher 1 ; Matthias Karg 4,8 ; Anders Barth 1,5,9 ; Claus A M Seidel 1 ; 1 Heinrich-Heine-University, Chair for Molecular Physical Chemistry, Düsseldorf, Germany 2 Heinrich-Heine-University, Institute for Physical Chemistry II, Düsseldorf, Germany 3 Abberior Instruments GmbH, Göttingen, Germany 4 Heinrich-Heine-University, Lehrstuhl für Kolloide und Nanooptik, Düsseldorf, Germany 5 Delft University of Technology, Department of Bionanoscience, Delft, The Netherlands 6 Scientific Volume Imaging, Hilversum, The Netherlands 7 Pulsar Photonics GmbH, Aachen, Germany 8 Martin-Luther-Universität, Physikalische Chemie funktionaler Polymere, Halle (Saale), Germany 9 PicoQuant GmbH, Berlin, Germany Super-resolution microscopy is an invaluable tool for minimally invasive studies of biomolecular assemblies. Recently, the lateral resolution reached the size of individual molecules.[1] Nevertheless, obtaining axial distances and hence, precise information on 3D orientation of structures remains challenging. Further, limitations of scanning speed may conceal fast dynamic exchange between different conformers of single molecules. We established an approach combining colocalization stimulated emission depletion (cSTED) microscopy with concurrent analysis of Förster resonance energy transfer (FRET) to identify heterogeneities in DNA systems.[2] We obtained precise lateral and axial distances using the Optical Pythagoras by comparing the localization distance between two fluorophores projected onto the image plane from cSTED to the Euclidean distance based on a structure model or obtained from concurrent FRET analysis. Taking full advantage of the synergy of joined STED localization and multiparameter FRET spectroscopy, we simultaneously localize donor and acceptor dyes of single FRET pairs, readily yielding sample orientation in 3D, with sub-nanometer localization precision for individual dyes. Thus, scanning 1x1 µm in a few seconds, we reached an average distance precision of 1.4 nm for a donor and acceptor label coupled to a single molecule. Studying immobilized dsDNA rulers, singly-bound to the surface, we measure a mean inclination angle of 64°, while for dsDNA rulers bound to the surface from both ends, we found an angle of 34°. The latter inclination angle correlates well with the local roughness of the surface found by AFM measurements. Using this knowledge, we provide a versatile workflow for performing FRET nanoscopy measurements down to the level of single particles, which also allows us to map conformational exchange within the 3D structure of DNA four-way junctions (Holliday junctions).References[1] Hell, S. W. et al, Science 2017, 355, 606-612.[2] Budde, J.- H. et al., arXiv preprint, 2022, doi.org/10.48550/arXiv.2108.00024

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