Scanning Electron Microscopy


Scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM), has been utilized to determine how the surface structure of mitotic chromosomes is related to the organization of the 30 nm chromosomal fibers. SEM revealed the surfaces of isolated, HeLa cell chromosomes to possess a knobby substructure with chromosomes prepared for EM in buffers containing 0.5-1.5 mM Mg2+. These projections had substantially greater widths (65-70 nm) than the underlying chromatin fibers. Reducing the Mg ion concentration to 0.05-0.15 mM resulted in the further expansion of the chromosomes, which flattened the chromosomes for SEM so the fibers became the dominant feature of the micrographs. The surface protuberances are interpreted as representing the peripheral tips of radial chromatin loops.

The same procedure of slightly expanding chromosomes by decreasing the Mg2+ concentration in resuspension buffer was also utilized in a TEM, serial sectioning study. Longitudinal sections close to the central chromatid axis showed radially oriented fibers within the planes of the sections. This was replaced by a dot pattern when the longitudinal sections grazed the periphery of the chromatid. Transverse sections displayed more clearly the radial orientation of the fibers.

A consistent picture emerges from applying SEM and TEM that supports the "radial loop" model for the primary mode of organization of chromatin fibers in metaphase chromosomes.

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