Biology subjects

Schuerstedt, J.

Publications and source records attributed to Schuerstedt, J..

3 recordsLinked to original sources

bioRxiv2024

Hydrogen peroxide damage to scavenging function and ultrastructure of liver sinusoidal endothelial cells is prevented by n-acetyl-cysteine but not GSH

Reactive oxygen species (ROS) are prevalent in the liver during intoxication, infection, inflammation, and ageing. Changes in liver sinusoidal endothelial cells (LSECs) are associated with various liver diseases. We investigated how oxidative stress induced by H2O2 affects isolated rat LSECs at different concentrations (0.5-1000{micro}M) and exposure times (10-120 min). Our findings show that H2O2 exposure affects several LSEC functions in a dose- and time-dependent manner: (1) cell viability, reducing potential, and scavenging function decreased as H2O2 concentration and exposure time increased; (2) intracellular ROS levels rose with higher H2O2 concentrations; (3) fenestrations exhibited a dynamic response, initially closing but partially reopening at H2O2 concentrations above 100{micro}M after about 1 h; (4) scavenging function was affected after just 10 min of exposure, with the impact being irreversible and primarily affecting degradation rather than receptor-mediated uptake; (5) the tubulin network was disrupted in high H2O2 concentration while the actin cytoskeleton appears to remain largely intact. Finally, we found that reducing agents and thiol donors such as N-Acetyl Cysteine (NAC) and Glutathione (GSH) could protect cells from ROS-induced damage but could not reverse existing damage. Pretreatment with NAC, but not GSH, reduced the negative effects of ROS exposure suggesting that LSEC does not store an excess amount of GSH but rather can readily produce it in the occurrence of oxidative stress conditions. The observed thresholds in dose and time-dependent changes as well as the treatments with NAC/GSH confirm the existence of ROS depleting system in LSEC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/609175v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1f25779org.highwire.dtl.DTLVardef@14cda58org.highwire.dtl.DTLVardef@92dea4org.highwire.dtl.DTLVardef@56e97c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIROS by H2O2 irreversibly depletes LSEC endocytic/scavenging function in vitro C_LIO_LIH2O2 exposure causes dynamic, dose-dependent defenestration of LSEC within 0.5 h C_LIO_LIPartial refenestration can occur after about 1h of exposure to H2O2 C_LIO_LINAC/GSH mitigate H2O2-induced ROS effects in LSEC C_LIO_LILSEC do not store excess GSH but produce GSH when exposed to oxidative stress C_LI

cell biology↗

bioRxiv2023

High-speed TIRF and 2D super-resolution structured illumination microscopy with large field of view based on fiber optic components

Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fast, and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high, and the physical size of the instruments based on the implementation of their optics is still rather large. Here, we present our latest results towards realizing a new generation of compact, cost-efficient, and high-speed SR-SIM instruments. Tight integration of the fiber-based structured illumination microscope capable of multi-color 2D- and TIRF-SIM imaging, allows us to demonstrate SR-SIM with a field of view of up to 150 x 150 m2 and imaging rates of up to 44 Hz while maintaining highest spatiotemporal resolution of less than 100 nm. We discuss the overall integration of optics, electronics, and software that allowed us to achieve this, and then present the fiberSIM imaging capabilities by visualizing the intracellular structure of rat liver sinusoidal endothelial cells, in particular by resolving the structure of their trans-cellular nanopores called fenestrations.

biophysics↗

bioRxiv2022

Dual-color single molecule localization microscopy on transparent polymer waveguide chips

Photonic waveguide chips offer near-field excitation of biological samples, which enables cost-effective, large field-of-view super-resolution microscopy without the need for high numerical aperture (NA) objective lenses. Single molecule localization based super-resolution microscopy that requires high illumination intensities is currently limited to solid state photonic waveguide chips composed of hard-coated, high NA planar waveguides deposited on opaque substrates. These platforms do not permit epi-detection of fluorescence through the substrate, which limits the use of photonic waveguide chips to the upright configuration. Additionally, the detection efficiency is reduced because the majority of the fluorescence emission is directed towards the high refractive index substrate. A low cost waveguide chip based on a polymer core material deposited on common #1.5 coverslips that is easy to produce was recently demonstrated. Here, a platform that is capable of performing single-molecule localization microscopy (SMLM) of biological samples using polymer-based photonic waveguide chips is presented, enabling super-solution microscopy in the inverted microscope configuration. Super-resolved imaging of two different structures of the cytoskeleton in primary liver sinusoidal endothelial cells (LSECs) by two popular SMLM methods, dSTORM and DNA-PAINT, down to 23 nm is demonstrated.

biophysics↗