Three-dimensional organization of chromatin fibers in situ examined by EM tomography
Electron tomography is being used to understand the 3D organization of chromatin in situ. As demonstrated previously, the nuclei of Patiria miniata (starfish) sperm contain particularly well-defined chromatin fibers. These studies are being extended through the analysis of 3D reconstructions of material embedded at low temperature in Lowicryl K11M and contrasted with osmium ammine-B, which preferentially stains nucleic acids. Tilt series of sections were recorded at 150KV, over an angular range of +/−75° and tilt increment of 2.5° using a Philips EM430. Image data were collected directly using a 1024x1024 CCD array with 2x2 binning to give a final pixel size of 1.3nm. Gold beads deposited on the sections were used for alignment, and reconstruction was by weighted back projection. Six volumes totalling 0.48um and containing numerous chromatin fibers have been examined utilizing Voxel View (Vital Images, Fairfield Iowa) software running on a Silicon Graphics Iris 4D workstation.