Explore the vast range of titles available.

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process. Neutrophilic granulocytes release their own DNA (NETosis) as neutrophil extracellular traps to capture pathogens. Here, the authors use time-resolved fluorescence and atomic force microscopy and reveal that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status.

Light microscopy has become an indispensable tool for the life sciences, as it enables the rapid acquisition of three-dimensional images from the interior of living cells/tissues. Over the last decades, super-resolution light microscopy techniques have been developed, which allow a resolution up to an order of magnitude higher than that of conventional light microscopy. Those techniques require labelling of cellular structures with fluorescent probes exhibiting specific properties, which are supplied from outside and therefore have to surpass cell membranes. Currently, major efforts are undertaken to develop probes which can surpass cell membranes and exhibit the photophysical properties required for super-resolution imaging. However, the process of probe development is still based on a tedious and time consuming manual screening. An accurate computer based model that enables the prediction of the cell permeability based on their chemical structure would therefore be an invaluable asset for the development of fluorescent probes. Unfortunately, current models, which are based on multiple molecular descriptors, are not well suited for this task as they require high effort in the usage and exhibit moderate accuracy in their prediction. Here, we present a novel fragment based lipophilicity descriptor DeepFL-LogP, which was developed on the basis of a deep neural network. DeepFL-LogP exhibits excellent correlation with the experimental partition coefficient reference data (R2 = 0.892 and MSE = 0.359) of drug-like substances. Further a simple threshold permeability model on the basis of this descriptor allows to categorize the permeability of fluorescent probes with 96% accuracy. This novel descriptor is expected to largely simplify and speed up the development process for novel cell permeable fluorophores.

A fluorescence microscope relying entirely on focused light allows the generation of spherical focal fluorescence spots much smaller than the wavelength of light. This development, termed isoSTED, overcomes the resolution limitation imposed by the diffraction of light and permits three-dimensional nanoscale imaging inside cells with common fluorophores. The resolution of any linear imaging system is given by its point spread function (PSF) that quantifies the blur of an object point in the image. The sharper the PSF, the better the resolution is. In standard fluorescence microscopy, however, diffraction dictates a PSF with a cigar-shaped main maximum, called the focal spot, which extends over at least half the wavelength of light ( λ = 400–700 nm) in the focal plane and > λ along the optical axis ( z ). Although concepts have been developed to sharpen the focal spot both laterally and axially, none of them has reached their ultimate goal: a spherical spot that can be arbitrarily downscaled in size. Here we introduce a fluorescence microscope that creates nearly spherical focal spots of 40–45 nm ( λ /16) in diameter. Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub- λ –sized organelles.

Analyzing the first and higher-order moments of the diffraction spot of a 4Pi fluorescence detection scheme facilitates two-color, three-dimensional super-resolution microscopy with ~6 nm axial and ~8–23 nm lateral resolution in a layer ~650 nm thick. We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.

The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.

Sister chromatid cohesion, mediated by cohesin and regulated by Sororin, is essential for chromosome segregation. In mammalian cells, cohesion establishment and Sororin recruitment to chromatin‐bound cohesin depends on the acetyltransferases Esco1 and Esco2. Mutations in Esco2 cause Roberts syndrome, a developmental disease in which mitotic chromosomes have a ‘railroad’ track morphology. Here, we show that Esco2 deficiency leads to termination of mouse development at pre‐ and post‐implantation stages, indicating that Esco2 functions non‐redundantly with Esco1. Esco2 is transiently expressed during S‐phase when it localizes to pericentric heterochromatin (PCH). In interphase, Esco2 depletion leads to a reduction in cohesin acetylation and Sororin recruitment to chromatin. In early mitosis, Esco2 deficiency causes changes in the chromosomal localization of cohesin and its protector Sgo1. Our results suggest that Esco2 is needed for cohesin acetylation in PCH and that this modification is required for the proper distribution of cohesin on mitotic chromosomes and for centromeric cohesion. Genetic deletion of murine Esco2 (an acetylase known to establish cohesin during S‐phase and genetically linked to Roberts syndrome) results in embryonic lethality that can be molecularly linked to novel and specific functions of Esco2 at pericentric heterochromatin.

Li et al . (Research Articles, 28 August 2015, aab3500) purport to present solutions to long-standing challenges in live-cell microscopy, reporting relatively fast acquisition times in conjunction with improved image resolution. We question the methods’ reliability to visualize specimen features at sub–100-nanometer scales, because the mandatory mathematical processing of the recorded data leads to artifacts that are either difficult or impossible to disentangle from real features. We are also concerned about the chosen approach of subjectively comparing images from different super-resolution methods, as opposed to using quantitative measures.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

By introducing beam-scanning multifocal multiphoton 4Pi-confocal microscopy, we have attained fast fluorescence imaging of live cells with axial super resolution. Rapid scanning of up to 64 pairs of interfering high-angle fields and subsequent confocal detection enabled us to perform three to five times finer optical sectioning than confocal microscopy. In conjunction with nonlinear image restoration, we demonstrate, to our knowledge for the first time, three-dimensional imaging of live eukaryotic cells at an equilateral resolution of ≈100 nm. This imaging mode allowed us to reveal the morphology and size of the green fluorescent protein-labeled mitochondrial compartment of live Saccharomyces cerevisiae (bakers' yeast) growing on different carbon sources. Our studies show that mitochondria of cells grown on medium containing glycerol as the only carbon source, as opposed to glucose-grown cells, exhibit a strongly branched tubular reticulum. We determine the average tubular diameter and find that it increases from 339 ± 5 nm to 360 ± 4 nm when changing from glucose to glycerol, that is, from a fermentable to a nonfermentable carbon source. Moreover, this change is associated with a 2.8-fold increase of the surface of the reticulum, resulting in an average increase in volume of the mitochondrial compartment by a factor of 3.0 ± 0.2.