The argyrophilic proteins of the nucleolar organizer region (Ag-NOR proteins) were specifically localized at the optical level with a modified one-step silver technique performed at 20 degrees C. This method was applied to various materials including cells in smears, chromosomes, semi-thin sections of plastic-embedded cells and sections of paraffin-embedded human pathological tissues. In order to improve the visualization of the silver deposits we tested various modes of imaging, including bright-field, Nomarski contrast, reflected light and combined Nomarski contrast with reflected light. The use of Nomarski contrast is useful to define precisely the phases of mitosis. The use of reflected light, which is based on the ability of silver to reflect incident light specifically, gives images with an improved resolution compared to bright-field.

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60 -80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250-to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

We have used the highly sensitive in situ terminal deoxynucleotidyl transferase method, applied to ultrathin sections, to investigate the location of DNA within nucleoli of various animal cells. In all the nucleoli studied, intense labelling is revealed over the peri- and intranucleolar condensed chromatin. Gold particles are also consistently found over the fibrillar centres, especially at their periphery, namely in the border area between the fibrillar centres and the dense fibrillar component, whereas the dense fibrillar component itself seems to be free of label in nucleoli in which these two compartments can be distinguished. We conclude that, in transcriptionally active nucleoli of this type, DNA is a characteristic constituent of the fibrillar centres, distinguishing them functionally from the dense fibrillar component. Some nucleoli exhibit neither fibrillar centres nor a dense fibrillar component, but have a single, albeit heterogeneous accumulation of fibrillar material; gold particles are consistently seen over some parts of this fibrillar compartment. This suggests that certain parts of the fibrillar material are functionally similar to the fibrillar centres of those nucleoli that possess them.

can be visualized through thick sections (several micro-Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3-8pm) of Epon-embedded cells at high tilt angles (-50"; + 50"). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-pm-thick section the values of thinning and shrinkage under the beam are 35 to 10% respectively. At the electron density used in these experiments ( 1 0 0 e -/~~/ s ) it is estimated that these modifications of the section stabilized in less than 10min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100nm for objects localized near the lower side of 4-pm-thick sections with a spot-size of 5.6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 pm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.