Three‐dimensional structured illumination microscopy (3D‐SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ∼100 nm range. We present first results and assess the potential using 3D‐SIM in combination with 3D fluorescence in situ hybridization (3D‐FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI‐stained DNA in nuclei before and after (...) 3D‐FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship ofXist‐RNA within the inactive X‐territory. These examples demonstrate that an appropriately adapted 3D‐FISH/3D‐SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D‐SIM yields new insights into the functional nuclear organization. (shrink)

This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less‐compacted (...) euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co‐aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments. (shrink)