Figure 1. Replication pattern of chromatin from a neuroblastoma cell line after double-pulse labeling during early and mid-S phase with nucleotides conjugated with different fluorochromes. (a) Light optical section through the nucleus fixed after double-pulse labeling. The nucleus shows the typical distribution pattern of early-replicating chromatin expanding in the nuclear interior and of mid-replicating chromatin concentrated at the nuclear periphery and around nucleoli. (b) Light optical section through a double-labeled nucleus fixed after three postlabeling cell cycles. The majority of nuclear chromatin remains unlabeled; the labeled chromatin shows polarized chromosome territories with typical early- and mid-replication patterns. (c) The same nucleus as shown in (b) after DNA counterstaining. (d) Metaphase spread prepared from double-labeled cells demonstrating distinct replication banding pattern of chromosomes. Scale bar for (a)–(c) is 5 m.

Figure 2. Distribution of territories from some large and small chromosomes after 3D FISH in nuclei of two cell types. The territories of small chromosomes are painted with a probe set specific for chromosomes 17–20; the territories of the large chromosomes are painted with a probe set specific for chromosomes 1–5. (Image courtesy of Alessandro Brero.) (a) 3D reconstruction (surface rendering, Amira TGS software) of chromosome territories combined with a mid-plane optical section of the counterstained nucleus (shown in gray) from a human fibroblast. All territories of the small chromosomes are clustered in the nuclear interior, while the territories of the large chromosomes show a peripheral positioning. (b) 3D reconstructions (surface rendering, Amira TGS software) of large and small chromosome territories in two nuclei of human lymphocytes. Territories of the larger chromosomes show a peripheral position compared with the chromosome territory position of the smaller chromosomes. Note, however, the peripheral position of a fraction of painted chromosome territories (marked by an arrow) that probably represent the territory of chromosome 18 (compare Cremer et al., 2001 ). Scale bars are 5 m.

Figure 3. Representative 3D reconstructions (surface rendering, Amira TGS software) of chromosome territories after painting of chromatin homologous to human chromosome 18 and human chromosome 19 in lymphoblastoid nuclei from different primate species. The chromosome territories are shown together with a partial reconstruction of the DNA-counterstained nuclear border. (a) Human lymphoblastoid cell. Both territories of chromosome 19 are clustered in the nuclear interior without touching the nuclear border, and the territories of chromosome 18 show a peripheral localization. (b) Common marmoset (Callithrix jacchus) lymphoblastoid cell. In this species, chromosomes homologous to human chromosomes 18 and 19 are entirely conserved except for a segment of chromosome 18 that is translocated to the 8p human homolog. The intranuclear positioning of chromatin homologous to human chromosomes 18 and 19 reveals the same spatial distribution as observed in humans. (c) White-handed gibbon (Hylobates lar) lymphoblastoid cell. Compared with humans, this species shows a high degree of chromosome reshuffling. The human chromosome 18 homologous segment is associated with the 1p32q22 human homologous segment while the human chromosome19 homolog is fragmented into four parts, which contribute to three different gibbon chromosomes. Irrespective of these major chromosome rearrangements, human chromosome 18 homologous chromatin is located in proximity to the nuclear envelope, whereas human chromosome 19 homologous material is centrally located, occasionally forming one large cluster as shown in the figure. Scale bar for (a)–(c) is 5 m. (Image courtesy of Hideyuki Tanabe.)

Figure 4. Visualization of centromeres in nuclei from different cell types. (a, b) View into the x,z plane of cuts through 3D reconstructions (volume rendering, Amira TGS software) of the spherical nucleus of a human lymphocyte (G₀, a) and of the flat nucleus of a human fibroblast (G₀, b). Centromeres are clustered at the nuclear periphery. The centromeres were visualized by 3D FISH with a pancentromeric (alphoid) probe. (c) 3D reconstruction (surface rendering, Amira TGS software) of simultaneously visualized territories and centromeres from chromosome 20 in the nucleus of a human lymphocyte (G₀) combined with the partially reconstructed nuclear border. The centromeres are located at the nuclear periphery, while the chromatin bulk of the territories protrudes into the nuclear interior. (d–h) Mid-optical sections through nuclei of human stimulated lymphocytes with immunostained kinetochores at different stages of the cell cycle. Note that centromeres (marked by kinetochores) have peripheral locations in early S phase and G₀, while during early G₁, late S and G₂ stages they are often found more internally. Scale bars are 5 m.