We survey the history of resolution enhancement techniques in microscopy and their impact on current research in biomedicine. Often these techniques are labeled superresolution, or enhanced resolution microscopy, or light-optical nanoscopy. First, we introduce the development of diffraction theory in its relation to enhanced resolution; then we explore the foundations of resolution as expounded by the astronomers and the physicists and describe the conditions for which they apply. Then we elucidate Ernst Abbe’s theory of optical formation in the microscope, and its experimental verification and dissemination to the world wide microscope communities. Second, we describe and compare the early techniques that can enhance the resolution of the microscope. Third, we present the historical development of various techniques that substantially enhance the optical resolution of the light microscope. These enhanced resolution techniques in their modern form constitute an active area of research with seminal applications in biology and medicine. Our historical survey of the field of resolution enhancement uncovers many examples of reinvention, rediscovery, and independent invention and development of similar proposals, concepts, techniques, and instruments. Attribution of credit is therefore confounded by the fact that for understandable reasons authors stress the achievements from their own research groups and sometimes obfuscate their contributions and the prior art of others. In some cases, attribution of credit is also made more complex by the fact that long term developments are difficult to allocate to a specific individual because of the many mutual connections often existing between sometimes fiercely competing, sometimes strongly collaborating groups. Since applications in biology and medicine have been a major driving force in the development of resolution enhancing approaches, we focus on the contribution of enhanced resolution to these fields.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.

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.