Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

40-POS Board 20 ADVANCING THE PERFORMANCE LIMITS AND APPLICATION SPACE OF SELF HEALING ORGANIC FLUOROPHORES Maxwell Martin 1,2 ; Sukanta Bar 1,2 ; Rakesh Paul 1,2 ; Roger Altman 1,2 ; Scott Blanchard 1,2 ; 1 St. Jude Children's Research Hospital, Structural Biology, Memphis, TN, USA 2 St. Jude Children's Research Hospital, Chemical Biology & Therapeutics, Memphis, TN, USA Organic fluorophores are a mainstay of single-molecule FRET, owing to their brightness, wavelength tunability, and ease of site-specific conjugation. By coupling cyanine dyes to cyclooctatetraene (COT) — which accesses a low-lying, Baird-aromatic triplet state and so efficiently quenches the dye's own triplet — our team has engineered "self-healing" fluorophores that sustain high emission and deliver up to 100-fold larger photon budgets. These dyes span the visible spectrum, and the same design principle now extends from in vitro single-molecule assays to live-cell applications including voltage sensing and long-term mitochondrial imaging. Despite these gains, fluorophore performance remains a limiting factor, and varies sharply with the biomolecule under study: dyes that excel on nucleic acids can lose an order of magnitude in performance on proteins. Photophysical and chemical characterization traces this to a previously unrecognized, protein-specific decay pathway — hydrogen-atom transfer (HAT) from the peptide backbone to fluorophores occupying higher-order excited states (S ₙ or T ₙ ), which degrades cyanines and rhodamines alike. The liability grows precisely as experiments push toward higher time resolution, where the higher illumination intensities required drive greater S ₙ /T ₙ occupancy. By naming this mechanism, we expose a fundamental limit of even today's best dyes and define a concrete target for the next generation — positioning us to extend the reach of single-molecule FRET in vitro and, ultimately, in vivo.

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