Z-series images of mouse ES cells carrying DHFR BAC transgene array (clone 36) and differentiated into fibroblast-like cells. 3D FISH shows increase in number of lac operator foci (green) and change to a more chain / fiber like large-scale chromatin conformation for the BAC transgene array (red). DAPI staining is shown in blue. Lac operator sequences still segregate into foci but these are located within or peripheral to fiber-like segments visualized for the BAC FISH signal. Nonlinear folding of DNA sequences within linear large-scale chromatin "fiber" segments is implied by the appearance of these Lac operator repeat foci.
Formaldehyde fixed cells were imaged using an IMT-2 Olympus fluorescence microscope with 60x 1.4 NA objective lens. Z-series were recorded, and the slices processed by iterative constrained deconvolution. Pixel sizes are 0.074 microns (xy) and z-spacing is 0.2 microns. See also: P Sinclair et al. 2010 Dynamic plasticity of large scale chromatin structure revealed by self-assembly of engineered chromosome regions. J Cell Biol 109:761-776.
|Spatial Axis||Image Size||Pixel Size|