Application of a novel technique for observing internal ultrastructure of human chromosomes with known karyotype

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has been hampered due to the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. We used a method in which the metaphase chromosome spreads were prepared on a flexible thermoplastic membrane, ACLAR film. To assess chromosome identity, a diamidino-phenylindole (DAPI) staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then impregnated with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and did carry whole chromosome spreads. Using this method, TEM observation of chromosomes from normal human lymphocytes and an ovarian carcinoma cell line (A2780) allowed thorough examination of the internal ultrastructure of centromeres, telomeres, fragile sites and other chromosomal regions. Chromosomes from normal lymphocytes showed a smooth and thick electron dense boundary. On the other hand, chromosomes from the cancer cells revealed different architectures with an irregular boundary with patchy electron densities at different locations and less density of the inside area. In addition, a thin electron dense boundary and numerous extended chromatin loops at the periphery were discernible indicating loosening of the condensed chromatin boundary. Such transition to a less condensed chromatin boundary may improve its availability for regulatory proteins and transcriptional factors. These findings provide a basis for further analysis using more sophisticated technologies such as cryo-electron microscopy or rapid freezing by freeze substitution to address cell type specificity of ultrastructural chromosome morphology. Taken together, application of the present method to chromosome research is expected to contribute vastly to the basic understanding of chromosome architecture and provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.