DNA packaging via hierarchical chromatin structures revealed by live-cell 3D imaging
Single-molecule localization microscopy is a powerful superresolution imaging technique to study biological questions by visualizing subcellular fine structures with nanometer-scale precision. However, its application in live-cell imaging studies has been impeded by the paucity of self-blinking organic fluorophores that enable high spatiotemporal resolution and labeling/localization density at a moderate laser intensity. Herein, we report a self-blinking Si-rhodamine dye 6-HESiR with a suitably increased "ON" fraction and a fluorogenic self-blinking dsDNA probe 6-HoeHESiR as a powerful tool for 3D superresolution imaging of native chromatin in eukaryotes without the use of photoswitching buffer and high laser intensity. With the probe 6-HoeHESiR, 3D superresolution imaging of in vitro reconstituted nucleosomal arrays and chromatin fibers yielded results consistent with EM analysis. Similar euchromatin and heterochromatin structures were visualized in fixed and live cells with high spatiotemporal resolution and labeling density, providing the first live-cell evidence for a hierarchical model of chromatin organization. 3D imaging results obtained in the presence of selective inhibitors of histone deacetylases also corroborate chromatin fiber decompaction upon hyperacetylation of histones.