Figure 2. Working range of conventional and quasi-bulk single particle FRET.

A–C) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A. At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.