BLeaching In-cell Single-molecule burstS (BLISS) reveals dynamic fraction of HP1α clusters in chromocenters of undifferentiated embryonic stem cells

Confocal fluorescence microscopy measurements of dense cellular regions of interest (ROIs) in cells using fluorescence lifetime imaging microscopy (FLIM) provide detailed pixelated images. Yet, those pixels report ensemble- and time-averaged biomolecular data, due to the diffraction limit and acquisition times that are slower than typical biomolecular dynamics. The ability to acquire data on one biomolecule at a time within a given ROI can help recover some of the underlying biomolecular subpopulations that are otherwise masked out. Here, we present a simple approach to achieving single-biomolecule photon bursts in live cells, dubbed BLeaching In-cell Single-molecule burstS (BLISS), which does not necessarily require super-resolution modalities. Using BLISS, we show that mCherry-tagged heterochromatin protein 1 (mCherry-HP1) in chromocenters of undifferentiated mouse embryonic stem cells (ESCs), exhibit photon bursts with millisecond durations arising from dynamic clusters. Fluorescence lifetimes of these bursts are substantially lower than the pixel-wise averaged values observed in FLIM, attributed to higher density in HP1 clusters. These higher density clusters are observed primarily in undifferentiated ESCs, while two days post retinoic acid (RA)-induced differentiation (2d-RA), these bursts are rarely observed. Using BLISS, we also detect dynamic clusters of the chromocenter-associated protein, CENP-V, and the nucleolar protein, nucleolin; however, in both cases the fluorescence lifetimes of their clusters closely resemble the pixel-wise averaged values recovered from FLIM. In conclusion, we introduce BLISS as a method for revealing rare and dynamic subpopulation in dense ROIs that are otherwise hidden by averaging in diffraction-limited imaging techniques. Importantly, using BLISS we demonstrate that denser-than-average HP1 clusters are hyperdynamic.