Fig. 4
Visualization of internal chromosome accessibility with super resolution 3D-SIM. a Untreated metaphase cell showing DA between chromosome 17 homologs (left panel, circled) hybridized with single copy FISH probe within PMP22:IVS3 (2.32 kb). Probe depth spans 1.30 μm or 10 of 17 (middle panel, red boxes*) and 0.65 μm or 5 of 17 (right panel, red boxes*) optical sections within accessible and less accessible homologs, respectively. b Decondensed metaphase chromosomes (left panel, boxed) hybridized with same PMP22:IVS3 (2.32 kb) single copy probe exhibit equal accessibility to both homologs. Probe depth (10 of 17 and 11 of 17 sections) for each homolog spans 1.30 μm (middle panel) and 1.43 μm (right panel), respectively. Same cell line (GM06326) is used in (a) and (b). Crosshairs are over maximal fluorescence. Der 17 refers to derivative chromosome 17. This was used as a cytogenetic marker to distinguish parental homologs. c Scatterplot of individual cells showing differences in hybridized probe volume and depth for untreated and treated cells. Normalized mean differences in hybridized probe volume (Δμ = 0.730 μm3, circles) and depth (Δμ = 0.651 μm, squares) for different untreated cells (n = 10 cells) for genomic target (PMP22:IVS3) with DA. These were significantly greater (volume: p = 0.003, depth: p = 0.013; two-tailed t test) compared to the same genomic target post-treatment (indicated with squares) in which both alleles were accessible (volume: Δμ = 0.237 μm3, depth: Δμ = 0.238 μm, n = 9 cells). Single cell outliers (ymax or xmax) with ICRF-193 treatment did not affect p-value cut off (α = 0.05). Normalized probe volume and depth were not strongly correlated pre- (r = 0.559) and post-treatment (r = 0.164). *Left red box is position zero for all panels in (a) and (b)