Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

72-POS

Board 36

ULTRA-RESOLUTION SINGLE-MOLECULE FRET Lukas Whaley-Mayda 1 ; Quan Wang 1 ;

1 National Institutes of Health, Laboratory of Chemical Physics, NIDDK, Bethesda, MD, USA Measuring FRET in single molecules (smFRET) has revolutionized our ability to probe biomolecular processes through the direct visualization of structural dynamics and heterogeneity. Yet, the resolution of smFRET measurements – and hence the magnitude of accessible structure changes – is often far poorer than the theoretical limits set by photon counting, restricting applications primarily to large, nanometer-scale conformational changes. Here we investigate multiple experimental factors influencing smFRET resolution and seek its ultimate achievable limits. Through systematic comparisons on DNA rulers, we find that resolution in conventional surface-tethered modalities is limited by artifactual heterogeneity introduced to varying degrees by the detection method, dye choices and immobilization strategy. By removing the requirement of surface-tethering with an anti-Brownian electrokinetic trap (ABEL-FRET), we show that it is possible to recover the intrinsic photon-limited resolution while delivering large photon budgets, leading to “ultra-resolution” smFRET. Strikingly, we find that many commonly used dye pairs produce structured or heterogeneous FRET peaks, precluding ultra-resolution performance. In cases of static or slowly changing structures where large numbers of photons (~10^4) can be used to calculate transfer efficiency, ultra-resolution extends the reach of smFRET to probing structure changes at the angstrom scale. We demonstrate these capabilities by resolving lesion induced distortions to DNA duplex structure at the single-molecule level.

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