Biology subjects

Zens, B.

Publications and source records attributed to Zens, B..

4 recordsLinked to original sources

bioRxiv2025

Azimuthal Segment Imaging in cryo-STEM Tomography

Cryo-electron microscopy is transitioning from investigation of isolated macromolecules to in situ studies bridging the realms of structural and cellular biology. Newly available detector technologies enable unconventional contrast modes with particular advantages. Here we demonstrate application of quadrant diode detectors to visualize a range of biological specimens by cryo-Scanning Transmission Electron Tomography (cryo-STET). Theoretically, we decompose coherent contrast by parallax analysis to isolate phase and amplitude contributions in specimens too thick for energy-filtered TEM. We thereby expand the cryo-STEM toolchest to parallax-filtered bright field (pBF) and parallax-filtered integrated differential phase contrast ({pi}DPC) and demonstrate their advantages in tomography using T4-bacteriophages, whole cells, and cryo-lift-out lamellae of cellular multilayers. The results show significant improvements over traditional STEM modalities in a realm where conventional wide-field transmission EM imaging methods are not applicable. The commercial availability of such detectors and the relative ease and speed of image reconstruction should make this realm accessible to the broader community in life science EM and beyond. TeaserParallax-corrected cryo-STET imaging provides coherent phase and amplitude contrast of thick biological specimens.

cell biology↗

bioRxiv2025

Image-based 3D active sample stabilization on the nanometer scale for optical microscopy

Super-resolution microscopy often entails long acquisition times of minutes to hours. Since drifts during the acquisition adversely affect data quality, active sample stabilization is commonly used for some of these techniques to reach their full potential. While drifts in the lateral plane can often be corrected after acquisition, this is not always possible or may come with drawbacks. Therefore, it is appealing to stabilize sample position in three dimensions during acquisition. Various schemes for active sample stabilization have been demonstrated previously, with some reaching sub-nm stability in three dimensions. However, these high-performance implementations significantly added to the complexity of the hardware and/or sample preparation. Here, we present a scheme for active drift correction that delivers the nm-scale 3D stability demanded by state-of-the-art super-resolution techniques and is straightforward to implement. Using a refined algorithm that does not depend on sparse peaks typically provided by fiducial markers added to the sample, we stabilized our sample position to [~]1 nm in 3D using objective lenses both with high and low numerical aperture. Our implementation requires only the addition of a standard widefield imaging path and we provide an open-source control software with graphical user interface to facilitate easy adoption of the module. Finally, we demonstrate how this has the potential to enhance data collection for diffraction-limited and super-resolution imaging techniques using single-molecule localization microscopy and cryo-confocal imaging as showcases. Why it mattersSuper-resolution light microscopy has enabled the visualization of biological structures down to the nm-scale. However, uncorrected drifts during often extended acquisition times may adversely affect data quality. Active drift correction in three dimensions has achieved sub-nm stabilization, but state-of-the-art techniques come with considerable overhead on sample preparation and/or hardware. Here, we demonstrate an image-based stabilization scheme which allows for flexibility regarding structures used for stabilization and is straightforward to adopt. Using a maximally simple implementation, we stabilized the position of our sample to around 1 nm over extended acquisition times and demonstrated usefulness in two example imaging settings where sample drifts are critical, super-resolution single-molecule localization microscopy and confocal imaging at cryogenic temperatures.

biophysics↗

bioRxiv2023

Unveiling the ultrastructural landscape of native extracellular matrix via lift-out cryo-FIBSEM and cryo-ET

The extracellular matrix (ECM) is a highly hydrated, three-dimensional network composed of various macromolecules and signaling factors. It serves as a structural scaffold for cells and plays an essential role in the regulation of numerous cellular processes, including cell migration, adhesion, and proliferation. Despite its importance in metazoans, structural knowledge is rudimentary on how the components of the matrisome are secreted, remodeled, and interact with each other and with surrounding cells. Specifically, the exact molecular assembly of important ECM fibers, such as fibronectin fibrils, fibrillin microfibrils, or Collagen-VI filaments has remained enigmatic. This is largely due to methodological limitations in specimen preparation for conventional room-temperature electron microscopy (EM). To overcome these limitations, we have developed a cell culture-based 3D-ECM platform compatible with sample thinning by cryo-lift out focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET). Our workflow involves the implementation of cell-derived matrices (CDMs) grown on EM grids, resulting in a highly adaptable and versatile tool to closely mimic ECM environments. This allows us to visualize native ECM and its components for the first time in their fully hydrated, cellular context. Our data reveals an intricate network of ECM fibers and their positioning with respect to matrix-secreting cells. In addition to D-spaced collagen fibers, we visualize previously unresolved fibrous structures, and an amorphous matrix co-assembling in proximity to ECM fibers and delineating the boundary between ECM and empty extra-cellular space. Intra- and extracellular granules presumably represent assembly intermediates of the ECM. Our results add to the structural atlas of the ECM and provide novel insights into ECM secretion, assembly and maintenance.

cell biology↗

bioRxiv2020

3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.

cell biology↗