Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

THE “BLISS” OF OBSERVING SINGLE-MOLECULE BURSTS FROM CLUSTERS IN LIVE CELL BIOMOLECULAR CONDENSATE Eitan Lerner 1,2 ; Eran Meshorer 3,4 ; Eden Mishne 3 ; Khalil Joron 1 ; 1 The Alexander Silberman Institute for Life Sciences, The Faculty of Sciences, The Hebrew University of Jerusalem, Biological Chemistry, Jerusalem, Israel 2 The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusal, Israel 3 The Alexander Silberman Institute for Life Sciences, The Faculty of Sciences, The Hebrew University of Jerusalem, Genetics, Jerusalem, Israel 4 Edmond and Lilly Center for Brain Sciences (ELSC), The Faculty of Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel For many years, the dream was to collect fluorescence-based data from single biomolecules in live cells. Recently, this has been achieved. The desire is still to acquire fluorescence-based data one biomolecular assembly at a time using endogenously-expressed proteins tagged with canonically-used monomeric fluorescent proteins (FPs) in highly-concentrated cellular foci. Here, I present a rather simple approach, dubbed BLISS, for acquiring clusters of mCherry tagged heterochromatin protein 1 α (HP1 α ) within heterochromatin condensates in live embryonic stem cells. Through this approach, I will show how such mCherry-HP1 α clusters exhibit lower fluorescence lifetimes than smaller mCherry-HP1 α species do and lower than average fluorescence lifetimes in FLIM pixels, revealing the source for heterogeneity that is averaged out in diffraction-limited fluorescence microscopy. RECENT DEVELOPMENTS OF ABEL-FRET: RESOLUTION LIMITS, FAST DYNAMICS AND BIOPHYSICAL APPLICATIONS Quan Wang 1 ; 1 National Institutes of Health, Lab of Chemical Physics, Bethesda, MD, USA In the past 30 years, single-molecule FRET (smFRET) has become a powerful and versatile method to probe conformations and dynamics at the molecular scale. We recently developed ABEL-FRET, a smFRET modality that achieves seconds-long single-molecule observations and ultrahigh, shot-noise limited resolution of smFRET efficiency, all without surface tethering. Here I present several new developments of ABEL-FRET, including ultimate resolution limits, resolving peptide conformers, probing dynamics from nanoseconds to seconds and applications to biomolecular structure and dynamics. These advances further establish ABEL-FRET as an emerging platform with unique advantages.

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