Biology subjects

Schulte am Esch, J.

Publications and source records attributed to Schulte am Esch, J..

4 recordsLinked to original sources

bioRxiv2026

Super-Resolution Optical Sectioning Microscopy Visualizes Nanopores in the Plasma Membrane of Endothelial Cells in situ

The ultrastructure of endothelial cells (ECs) "in situ" is of great interest due to their involvement in many physiological processes. In some organs, these cells form transcellular pores or fenestrae, allowing for the rapid exchange of molecules between blood and interstitium. Despite their importance, no optical images of these dynamic morphological structures have yet been acquired in situ. Major obstacles to their in-situ imaging are the lack of specifical labels for fenestrae and their size well below the optical diffraction limit. Here, we report how we have overcome these challenges and managed to visualize the EC ultrastructure in situ in 25 {micro}m thick liver sections. To enable this, a lipophilic, fluorescent membrane dye was infused into the portal vein of murine livers to stain the sinusoidal ECs before the organ was harvested. Tissue sections were subsequently imaged using a novel, super-resolution optical-sectioning structured illumination microscope (OS-SIM), providing approx. 170 nm spatial resolution with significantly faster image acquisition compared to confocal microscopy.

biophysics↗

bioRxiv2026

Enhanced 2D structured illumination microscopy: super-resolution with optical sectioning and reduced reconstruction artifacts

Structured Illumination Microscopy (SIM) provides imaging with spatial super-resolution, as well as optical sectioning capability, without relying on specialized fluorescent dyes. 2D and 3D variants of this method exist, but most bespoke implementations are 2D-SIM, because it is easier to realize and modify than 3D-SIM. 2D-SIM systems, however, often experience reconstruction artifacts from out-of-focus contributions, especially when pushing for high lateral spatial resolution in thicker samples. We present enhanced 2D-SIM, an approach to 2D-SIM where both, coarse patterns optimized for removing out-of-focus background, and fine patterns optimized for resolution improvement beyond the diffraction limit are used. In combination, this achieves 2D-SIM reconstructions with high contrast, spatial super-resolution, and significantly reduced reconstruction artifacts. We present the theoretical framework of this technique, and provide enhanced 2D-SIM imaging results of liver sinusoidal endothelial cells stained with fluorophores emitting at visible and near-infrared wavelengths. Quantitative comparisons of power spectral distribution and image resolution are demonstrated.

biophysics↗

bioRxiv2025

Offsetting ROS-mediated arrest of endothelial fenestration dynamics permits long-term optical super-resolution- and AFM-imaging

Advances in cell biology demand methods that resolve the structure and dynamics of subcellular organelles in living cells. Live-cell super-resolution fluorescence microscopy meets this need but is constrained by phototoxicity, which disturbs cellular function and biases interpretation. Liver sinusoidal endothelial cells (LSECs), with their physiologically critical and highly dynamic fenestrations, represent a particularly challenging model system. We show that photoactivation-generated reactive oxygen species (ROS) are the primary cause of fenestration arrest during fluorescence imaging. Using three-dimensional structured illumination microscopy (3D SR-SIM), we systematically evaluated fluorophores and ROS scavengers to optimize imaging conditions. A combination of BioTracker staining and CO2-independent medium supplemented with N-acetylcysteine (NAC) preserved fenestration dynamics without altering fenestration number or size. Complementary atomic force microscopy (AFM) confirmed ROS-dependent impairment of fenestration dynamics and revealed nanomechanical changes upon illumination. These findings establish the mechanism underlying imaging-induced artefacts in LSECs and provide a broadly applicable strategy to extend live-cell super-resolution microscopy.

cell biology↗

bioRxiv2020

Simulating digital micromirror devices for patterning coherent excitation light in structured illumination microscopy

Digital micromirror devices (DMDs) are spatial light modulators that employ the electro-mechanical movement of miniaturized mirrors to steer and thus modulate the light reflected of a mirror array. Their wide availability, low cost and high speed make them a popular choice both in consumer electronics such as video projectors, and scientific applications such as microscopy. High-end fluorescence microscopy systems typically employ laser light sources, which by their nature provide coherent excitation light. In super-resolution microscopy applications that use light modulation, most notably structured illumination microscopy (SIM), the coherent nature of the excitation light becomes a requirement to achieve optimal interference pattern contrast. The universal combination of DMDs and coherent light sources, especially when working with multiple different wavelengths, is unfortunately not straight forward. The substructure of the tilted micromirror array gives rise to a blazed grating, which has to be understood and which must be taken into account when designing a DMD-based illumination system. Here, we present a set of simulation frameworks that explore the use of DMDs in conjunction with coherent light sources, motivated by their application in SIM, but which are generalizable to other light patterning applications. This framework provides all the tools to explore and compute DMD-based diffraction effects and to simulate possible system alignment configurations computationally, which simplifies the system design process and provides guidance for setting up DMD-based microscopes.

biophysics↗