Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

28-POS Board 14 BLEACHING IN-CELL SINGLE-MOLECULE BURSTS (BLISS) UNCOVERS DYNAMIC HP1A SUBPOPULATIONS IN DENSE CHROMOCENTERS OF PLURIPOTENT EMBRYONIC STEM CELLS Khalil Joron 1 ; Eden Mishne 2 ; Eran Meshorer 2,3 ; Eitan Lerner 1,4 ; 1 The Alexander Silberman Institute of Life Sciences at The Hebrew University of Jerusalem, Department of Biological Chemistry, Jerusalem, Israel 2 The Alexander Silberman Institute of Life Sciences at The Hebrew University of Jerusalem, Department of Genetics, Jerusalem, Israel 3 Edmond and Lily Center for Brain Sciences (ELSC) at The Hebrew University of Jerusalem, Jerusalem, Israel 4 The Center for Nanoscience and Nanotechnology at The Hebrew University of Jerusalem, Jerusalem, Israel In dense cellular regions of interest (ROIs), fluorescence lifetime imaging microscopy (FLIM) reports ensemble- and time-averaged biomolecular information, due to diffraction-limited pixels. As a result, these pixels contain multiple molecules and acquisition rates that are slower than biomolecular dynamics, thereby masking rare biomolecular subpopulations. Acquiring data one biomolecule at a time within a given ROI can reveal these otherwise hidden biomolecular assemblies. Here, we leverage the balance between continuous photobleaching and molecular diffusion to reduce fluorescence to a steady-state level as close to the detector background as possible. Under these conditions, individual biomolecules contribute predominantly to the background rate, whereas brighter biomolecular clusters generate detectable single-molecule photon bursts resembling those observed in confocal-based single-molecule spectroscopy. We term this approach BLeaching In-cell Single-molecule burstS (BLISS). Using BLISS, we measured the fluorescence lifetimes of endogenously expressed mCherry-tagged heterochromatin protein 1 α (HP1 α ) in heterochromatin condensates of live mouse embryonic stem cells (ESCs), known as chromocenters. Previous FLIM studies showed that reduction in mCherry lifetime reports increase in local molecular density and revealed heterogeneity within HP1 α condensates, but the molecular origin of this heterogeneity remained elusive. BLISS resolves the fluorescence lifetime distribution of individual diffusing HP1 α clusters, revealing a rare subpopulation with shorter lifetimes relative to the pixel-averaged FLIM values, indicative of higher local molecular density. We show that these dense, dynamic clusters are abundant in pluripotent ESCs but nearly absent two days post retinoic acid induced differentiation. Using BLISS we also detect dynamic clusters of the chromocenter-associated protein CENP-V, and the nucleolus-associated protein, nucleolin; however, in both cases their fluorescence lifetimes closely match the pixel-averaged FLIM values, indicating that these clusters exhibit densities similar to the average densities per pixel, unlike HP1 α . These results demonstrate the capability of BLISS to resolve dense molecular subpopulations that are masked by averaging in diffraction limited fluorescence imaging.

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