Fig. 3: Probing biomolecular structures in living cells with STARSS.

a, Live human U2OS cells expressing rsEGFP2 targeted to the mitochondrial outer membrane (OM) with different linker sizes were measured using STARSS method 1. A short rigid helical linker (blue, integrated over n = 9 cells) resulted in higher anisotropy values compared with a longer flexible linker (orange, integrated over n = 7 cells). b, Cartoon of histone H2B fused with rsEGFP2 via a prolonged terminal alpha-helix (upper left) as a unit of chromatin filaments of size of ~30 nm (bottom). c, Anisotropy recorded in the rigid and flexible constructs of H2B-rsEGFP2 in U2OS cells with STARSS method 1, in live cells and fixed cells (integrated over n = 3 cells). d, Representative fluorescence intensity image and segmentation map of nucleoli/nucleus (left, scale bar 4 μm) used to split the anisotropy measured with STARSS method 1 in blue and orange datasets (right, integrated over n = 8 cells). e, Anisotropy decays recorded with STARSS method 1 in HeLa cells expressing rigid H2B-rsEGFP2 for interphase chromatin (integrated over n = 8 cells), mitotic chromosomes (integrated over n = 18 cells), treatment of interphase chromatin with TSA (integrated over n = 8 cells) and treatment and recovery of interphase chromatin for ATP depletion (ATPd, both integrated over n = 8 cells). f, Fluorescence count maps (left) and fluorescence anisotropy maps (right) for two representative fields of view of chromatin in an interphase nucleus after ATPd (top), and mitotic chromosomes (bottom). Scale bars, 4 μm. In all relevant panels, error bars and shaded regions of anisotropy values are 95% confidence intervals evaluated from detector noise according to Supplementary Information 20.