Biology subjects

Hübner, W.

Publications and source records attributed to Hübner, W..

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↗

bioRxiv2020

Recruitment of Env to the HIV-1 T cell virological synapse by targeted and sustained Env recycling

HIV-1 infection is enhanced by cell-cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Using surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, we observed targeted accumulation and sustained recycling of Env between the endocytic recycling compartment (ERC) and the VS. We observed dynamic exchange of Env at the VS while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse indicate that retention of Env is not likely to be maintained by entrapment into an immobile Gag lattice or through immobilizing interactions with CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus.

microbiology↗

bioRxiv2019

DMD-based super-resolution structured illumination microscopy visualizes live cell dynamics at high speed and low cost

Structured illumination microscopy (SIM) is among the most widely used super-resolution fluorescence microscopy techniques for visualizing the dynamics of cellular organelles, such as mitochondria, the endoplasmic reticulum, or the cytoskeleton. In its most wide-spread implementation, SIM relies on the creation of an interference pattern at the diffraction limit using the coherent addition of laser beams created by a diffraction pattern.\n\nSpatial light modulators based on liquid crystal displays allow SIM micro-scopes to run at image rates of up to hundreds of super-resolved images per second. Digital micromirror devices are another natural choice for creating interference-based SIM patterns, but are not used to their fullest potential because of the blazed grating effect. This effect arises due to the fixed angles between which the mirrors can be switched, creating a sawtooth arrangement of mirrors and thus leading to a change in the intensity distribution of the diffracted beams. This results in SIM patterns with varying modulation contrast which are prone to reconstruction artifacts.\n\nWe have carefully studied the blazed grating effect of DMDs by simulations, varying a range of parameters and compared the simulation results with experiments. This allowed us to identify settings which result in very high modulation contrast across all angles and phases required to generate 2-beam SIM pattern. The use of inexpensive industry-grade CMOS cameras as well as low-cost lasers enabled us to construct a cost-effective, high-speed SIM system. Reconstruction of the super-resolved SIM images is achieved on a recently demonstrated parallel-computing platform, which allowed us to visualize living cells with super-resolution at multiple reconstructed frames per second in real time. We demonstrate the versatility of this new platform by imaging cellular organelle dynamics based on live-cell fluorescent stains as well as with fluorescent protein stained samples.

biophysics↗

bioRxiv2019

High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work we present a multi-plane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multi-plane image splitting prism combined with high-speed SR-SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally super-resolved three-dimensional image stack. We demonstrate the performance of multi-plane SR-SIM by applying this instrument to the dynamics of live mitochondrial network.

biophysics↗