Figure 5

Visualization of metaphase chromosome differential accessibility in 2- and 3-dimensions. A. Epifluorescence image of metaphase cell hybridized with HERC2 single copy probe (1.81 kb) shows a DA pattern. Chromosome 15 homologs are magnified. 3D structured illumination microscopy of hybridized probe volume (panel B) and probe depth (panel C) for the magnified homologs in panel A are presented. B. The left homolog with greater accessibility contains fluorescence embedded within the chromosome and protrudes above the surface. In contrast, the right homolog with less accessibility has a much smaller volume of hybridized probe fluorescence and is mainly embedded within the chromosome. Reconstructed volume view in the left homolog was generated by rotating it clockwise about the z-axis (see orientation schematic). Volume view in the right homolog was generated by up-righting it (arrow 1) and turning it clockwise (arrow 2) (see schematic). C. Crosshairs are centered over the maximal fluorescent intensity projection along the XY, XZ and YZ axes for each chromosome 15 homolog, and highlight differences in chromatin accessibility. The axial projection (depth) of the probe fluorescence spans 18 of 21 0.1 μm reconstructed optical sections (white rectangles delineate boundaries along the z axis) in the left more accessible homolog; and only 12 of 21 reconstructed optical sections in the right homolog (white rectangles).