3-DIMENSIONAL COLOCALIZATION OF NUCLEOLAR ARGYROPHILIC COMPONENTS ANDDNA IN CELL-NUCLEI BY CONFOCAL MICROSCOPY

Silver dots deposited specifically on proteins of the nucleolar organi zer regions (Ag-NOR proteins) after a one-step silver staining techniq ue were visualized in cells in culture, in cells in smears, and in tis sue sections, with a scanning laser confocal microscope working in the reflectance mode. After specific labeling of DNA with the fluorescent dye chromomycin Ag, DNA and silver dots could be observed either indi vidually or simultaneously. Therefore, it was possible to study the th ree-dimensional organization of nucleolar silver-stained structures re lative to DNA with a high X, Y, and Z resolution. Our results showed t hat the argyrophilic components are organized as a twisted necklace st ructure within interphase nucleoli of cells in culture. We also demons trated a striking three-dimensional symmetric disposition of NORs with in the two sets of chromosomes in telophase cells. Similar results wer e obtained for cells in smears, although their three-dimensional organ ization was somewhat disturbed due to air-drying. We also demonstrated that silver dots cannot be visualized in the reflectance mode within sections of paraffin-embedded tissues. However, their simultaneous dem onstration in non-confocal transmitted light, together with that of DN A in confocal mode, appeared very useful to study their localization w ithin nuclei and mitotic chromosomes.