By Claudio Nicolini
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Extra resources for Chromatin Structure and Function: Molecular and Cellular Biophysical Methods
In order to visualize nucleosomal ultrastructure, a micrograph of isolated monomer nucleosomes (Figure 2) is presented. Again, darkfield is used to achieve contrast. The clarity of detail is due, in part, to the high magnification (200,000X) at which the micrograph ,,,as made and, in part, to the low concentration of stain used. The similarity of the particles in the chromosomal fibers of Figure 1 and the nucleosomes (VI) isolated after micrococcal nuclease digestion in Figure 2 is demonstrated.
These are difficult questions, whose 34 A. L. DLiNS answers change as new techniques are devised. At present, however, we can suggest and justify criteria adopted within our laboratory for preparing chromatin specimens: 1. Chromatin tends to form gelatinous fibrillar aggregates, which should be avoided because the underlying substructure of the fibers is obscured or altered. It is especially helpful to use freshly isolated nuclei or chromatin. Frozen cells, frozen nuclei or frozen chromatin all tend to form gelatinous aggregates.
The nucleosomes, cannot be clearly visualized in darkfield microscopy. This is due, in part, to the extra electron density of th e negative stain surrounding the fiber and, in part, to the superimposition of nucleosomes on opposite sides of the thick chromatin fiber. This micrograph is slightly underfocus, as can be seen by the increased contrast and the crisp symmetric background grain. 38 A. L. DLiNS Figure 3. A bright field electron micrograph of a 20-30 nm chromatin fiber at the periphery of a chicken erythrocyte nucleus.