Joron, K.; Mishne, E.; Meshorer, E.; Lerner, E.

Fluorescence imaging of dense cellular regions of interest (ROIs) in cells using fluorescence microscopy provides detailed images with pixels that report ensemble- and time-averaged biomolecular data, due to the diffraction limit when super-resolution modalities are not used and acquisition times are slower than typical biomolecular mobilities. The fluorescently-tagged biomolecules that are undergoing imaging can be more heterogeneous and dynamic, all within the dimensions of a single acquired image pixel. The ability to acquire data one biomolecule at a time within a given ROI can help recover some of the underlying biomolecular subpopulations that are otherwise averaged out. In this work, we present a relatively simple approach to achieving single-biomolecule photon bursts, BLeaching In-cell Single-molecule burstS (BLISS). We reveal millisecond photon bursts arising from clusters of mCherry-tagged heterochromatin protein 1 (mCherry-HP1) within heterochromatin biomolecular condensates in undifferentiated mouse embryonic stem cells (ESCs). Fluorescence lifetimes of these bursts are substantially lower than the averaged-out values observed per pixel in fluorescence lifetime imaging microscopy (FLIM), attributed to higher density in mCherry-HP1 clusters. These higher density clusters are observed primarily in undifferentiated ESCs. Two days after retinoic acid (RA) induction of differentiation, these bursts are rarely observed. In summary, using BLISS, we revealed a rare subpopulation of dense mCherry-HP1 clusters characterized by rapid, millisecond dynamics. These clusters are part of heterochromatin biomolecular condensates in ESCs at the pluripotent state, which would be otherwise averaged out in diffraction-limited fluorescence microscopy.