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Epigenetic dysregulation of chromatin is one of the hallmarks of cancer development and progression, and it is continuously investigated as a potential general bio-marker of this complex disease. One of the nuclear factors involved in gene regulation is the unique DEK protein—a histone chaperon modulating chromatin topology. DEK expression levels increase significantly from normal to cancer cells, hence raising the possibility of using DEK as a tumor marker. Although DEK is known to be implicated in epigenetic and transcriptional regulation, the details of these interactions and their relevance in cancer development remain largely elusive. In this work, we investigated the spatial correlation between the nuclear distribution of DEK and chromatin patterns—alongside breast cancer progression—leveraging image cross-correlation spectroscopy (ICCS) coupled with Proximity Ligation Assay (PLA) analysis. We performed our study on the model based on three well-established human breast cell lines to consider this tumor's heterogeneity (MCF10A, MCF7, and MDA-MB-231 cells). Our results show that overexpression of DEK correlates with the overall higher level of spatial proximity between DEK and histone marks corresponding to gene promoters regions (H3K9ac, H3K4me3), although it does not correlate with spatial proximity between DEK and gene enhancers (H3K27ac). Additionally, we observed that colocalizing fractions of DEK and histone marks are lower for the non-invasive cell subtype than for the highly invasive cell line (MDA-MB-231). Thus, this study suggests that the role of DEK on transcriptionally active chromatin regions varies depending on the subtype of the breast cancer cell line. [ABSTRACT FROM AUTHOR]

The well-known saying of 'Seeing is believing' became even more apt in biology when stimulated emission depletion (STED) nanoscopy was introduced in 1994 by the Nobel laureate S. Hell and coworkers. We presently stand at a juncture. Nanoscopy represented a revolution in fluorescence microscopy but now is a mature technique applied to many branches of biology, physics, chemistry, and materials science. We are currently looking ahead to the next generation of optical nanoscopes, to the new key player that will arise in the forthcoming years. This article gives an overview of the various cutting-edge implementations of STED nanoscopy and tries to shine a light into the future: imaging everything faster with unprecedented sensitivity and label-free. [ABSTRACT FROM AUTHOR]

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells. A known limitation of super-resolution STED microscopy is the need of high laser power which can cause photobleaching and phototoxicity. Here the authors further optimize this method and show that modulating STED intensity during acquisition results in an enhanced resolution and reduced background. [ABSTRACT FROM AUTHOR]