Super-resolution microscopy (SRM) is a fast-developing field that encompasses fluorescence imaging techniques with the capability to resolve objects below the classical diffraction limit of optical resolution. Acknowledged with the Nobel prize in 2014, numerous SRM methods have meanwhile evolved and are being widely applied in biomedical research, all with specific strengths and shortcomings. While some techniques are capable of nanometre-scale molecular resolution, others are geared towards volumetric three-dimensional multi-colour or fast live-cell imaging. In this editorial review, we pick on the latest trends in the field. We start with a brief historical overview of both conceptual and commercial developments. Next, we highlight important parameters for imaging successfully with a particular super-resolution modality. Finally, we discuss the importance of reproducibility and quality control and the significance of open-source tools in microscopy. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Significance: The structure of the pachytene chromosome is essential to understand how genetic recombination can happen during meiosis. Using single molecule localization microscopy with DNA staining, we show that chromatin is heavily constrained by defined periodic clusters along the synaptonemal complex (SC). Staining of various posttranslational histone modifications further reveals that the pachytene chromosome is associated with three distinct nanoscale compartments. Whereas chromatin associated with active transcription emanates both axially and radially in hair-like loop structures, the chromatin associated with repressed transcription follows periodic clusters close to the central axis of the SC. These findings suggest a model showing how chromatin and epigenetic modification patterns can be incorporated within the SC to shape the pachytene chromosome.

Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. ...

Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones. Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40–700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.