Nanoscale distribution of TLR4 on primary human macrophages stimulated with LPS and ATI

Toll-like receptor 4 (TLR4) plays a crucial role in the recognition of invading pathogens. Upon activation by lipopolysaccharides (LPS), TLR4 is recruited into specific membrane domains and dimerizes. In addition to LPS, TLR4 can be stimulated by wheat amylase-trypsin inhibitors (ATI). ATI are proteins associated with gluten containing grains, whose ingestion promotes intestinal and extraintestinal inflammation. However, the effect of ATI vs. LPS on the membrane distribution of TLR4 at the nanoscale has not been analyzed. In this study, we investigated the effect of LPS and ATI stimulation on the membrane distribution of TLR4 in primary human macrophages using single molecule localization m…

Screening of herbal extracts for TLR2- and TLR4-dependent anti-inflammatory effects.

Herbal extracts represent an ample source of natural compounds, with potential to be used in improving human health. There is a growing interest in using natural extracts as possible new treatment strategies for inflammatory diseases. We therefore aimed at identifying herbal extracts that affect inflammatory signaling pathways through toll-like receptors (TLRs), TLR2 and TLR4. Ninety-nine ethanolic extracts were screened in THP-1 monocytes and HeLa-TLR4 transfected reporter cells for their effects on stimulated TLR2 and TLR4 signaling pathways. The 28 identified anti-inflammatory extracts were tested in comparative assays of stimulated HEK-TLR2 and HEK-TLR4 transfected reporter cells to dif…

Back Cover: Nanographenes: Ultrastable, Switchable, and Bright Probes for Super‐Resolution Microscopy (Angew. Chem. Int. Ed. 1/2020)

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

Abstract Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and…

Nanographene: ultrastabile, schaltbare und helle Sonden für die hochauflösende Mikroskopie

Super-resolution binding activated localization microscopy through reversible change of DNA conformation

ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future app…

High-resolution deep view microscopy of cells and tissues

Abstract Methods, experimental setups and perspectives of three-dimensional deep view imaging microscopy of cell or tissue samples are reported. Preliminary biophysical and clinically relevant examples are presented.

Rücktitelbild: Nanographene: ultrastabile, schaltbare und helle Sonden für die hochauflösende Mikroskopie (Angew. Chem. 1/2020)

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses…

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of …

Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes.

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 ill…

Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.

Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density,…

Entering the Nano-Cosmos of the Cell by Means of Spatial Position Determination Microscopy (SPDM): Implications for Medical Diagnostics and Radiation Research

During the last 20 years fluorescence light microscopy has made an enormous progress towards fluorescence nanoscopy in order to elucidate the nanostructural organization of cellular machineries beyond classical limits of resolution in light microscopy. One of these novel techniques is Spatial Position Determination Microscopy (SPDM), an approach of molecular localization microscopy based on the application of specific fluorescence labelling of cellular structures by means of dyes that undergo reversible photobleaching resulting in blinking effects during image acquisition. This blinking allows spectral separation of individual molecules and thus precise localization and distances measuremen…

Nitration of Wheat Amylase Trypsin Inhibitors Increases Their Innate and Adaptive Immunostimulatory Potential

Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can mediate innate immunity via activation of the Toll-like receptor 4 (TLR4) on immune cells residing in the lamina propria, promoting intestinal, as well as extra-intestinal, inflammation. Inflammatory conditions can induce formation of peroxynitrite (ONOO-) and, thereby, endogenous protein nitration in the body. Moreover, air pollutants like ozone (O3) and nitrogen dioxide (NO2) can cause exogenous protein nitration in the environment. Both reaction pathways may lead to the nitration of ATI. To investigate if and how nitration mo…

Der Zellkern - eine Stadt in der Zelle

Gespeicherte genetische Information allein nutzt nichts, wenn sie nicht zur richtigen Zeit und am richtigen Ort abgerufen werden kann. Die Verpackung der DNA im Chromatin des Zellkerns und dessen dynamische, raum-zeitliche Anordnung haben einen entscheidenden Einfluss auf die Genexpression und weitere Funktionen des Zellkerns. In dieser Ubersichtsarbeit beschreiben die Verfasser die Entwicklung der experimentellen Zellkernarchitektur-Forschung und die damit einhergehenden Veranderungen der Vorstellungen zur funktionellen Organisation des Zellkerns.

Nanographenes: Ultrastable, Switchable, and Bright Probes for Super-Resolution Microscopy.

Abstract Super‐resolution fluorescence microscopy has enabled important breakthroughs in biology and materials science. Implementations such as single‐molecule localization microscopy (SMLM) and minimal emission fluxes (MINFLUX) microscopy in the localization mode exploit fluorophores that blink, i.e., switch on and off, stochastically. Here, we introduce nanographenes, namely large polycyclic aromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots, as a new class of fluorophores for super‐resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties: intrinsic blinking even in air, excellent fluorescence recovery, and sta…