3D-SIM-based DAPI intensity classification in the Barr body versus the entire nucleus of C2C12 cells. (A) Mid z-section of a DAPI-stained nucleus. The area below the dashed line illustrates the resolution level obtained by wide-field deconvolution microscopy, for comparison. Inset magnifications show the non-uniformly compacted structure of the Barr body resolvable with 3D-SIM (1) and an arbitrary autosomal region with CDCs (2). Scale bars: 5 μm, insets 1 μm. (B) X chromosome-specific painting (green) of Xi (left) and Xa territories (right) of the same nucleus in different z-sections. Note the high convergence between the painted Xi and the DAPI visualized Barr body (arrowheads). Scale bars: 2 μm, insets 1 μm. (C) 3D DAPI intensity classification exemplified for the nucleus shown in (A). Seven DAPI intensity classes displayed in false-color code ranging from class 1 (blue) representing pixels close to background intensity, largely representing the IC, up to class 7 (white) representing pixels with highest density, mainly associated with chromocenters. Framed areas of the Barr body (inset 1) and a representative autosomal region (inset 2) are shown on the right at resolution levels of 3D-SIM, deconvolution and conventional wide-field microscopy. The Xi territory pervaded by lower DAPI intensities becomes evident only at 3D-SIM resolution, whereas both wide-field and deconvolution microscopy imply a concentric increase of density in the Barr body. In the autosomal region, chromatin assigned to classes 2 to 3 lines compacted CDCs, represented by classes 4 to 6. (D) Left: average DAPI intensity classification profiles with standard deviations evaluated for entire nuclear volumes or the Barr body region only (dark grey bars). Right: over/underrepresentation of the average DAPI intensity class fraction sizes in the Barr body versus entire nuclear volumes (n = 12). Distribution differences on classes between Xi and entire nucleus P <0.001. 3D-SIM, three-dimensional structured illumination microscopy; CDC, chromatin domain cluster; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; IC, interchromatin compartment; Xa, active X chromosome; Xi, inactive X chromosome.