Biology subjects

de Beer, M.

Publications and source records attributed to de Beer, M..

5 recordsLinked to original sources

bioRxiv2026

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.

biophysics↗

bioRxiv2025

Overglycosylation introduces local changes in triple helix alignment in collagen type I fibril structure

Collagen fibrils constitute the structural scaffold of bone, and their hierarchical organization is central to biomineralization. While hydroxylation and glycosylation of lysine residues are well-known collagen post-translational modifications, their structural consequences remain poorly understood. Here we show that excess of glycosylation of hydroxylysine residues leads to significant alterations in the local packing of collagen molecules within the fibrils. By prolonging the enzymatic modification window during helix folding through the use of cyclosporin A, an increase in double glycosylation was observed at specific sites. These overglycosylated residues were pinpointed by mass spectrometry, while cryogenic TEM revealed fibrils with reduced diameters and distinct displacements of defined sub-bands within the D-period, without altering the overall periodicity. By mapping the modified residues onto the quarter-staggered model, we have been able to correlate site-specific glycosylation with sub-band shifts, linking chemical modification to supramolecular order. These results provide molecular-level evidence that collagen glycosylation is an active determinant of fibril structure. Such insights not only advance fundamental understanding of collagen assembly but could also illuminate mechanisms underlying bone fragility disorders, including osteogenesis imperfecta, that feature altered glycosylation.

biochemistry↗

bioRxiv2022

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

cell biology↗

bioRxiv2022

DNA uptake by cell wall-deficient bacteria reveals a putative ancient macromolecule uptake mechanism

Horizontal gene transfer in bacteria is widely believed to occur via three main mechanisms: conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. We present here a new mechanism of DNA uptake that is independent of canonical DNA uptake machineries and is used by bacteria that live without a cell wall. We show that the cell wall-deficient bacteria engulf extracellular material, whereby intracellular vesicles are formed, and DNA is internalized. This mechanism is not specific to DNA, and allows uptake of other macromolecules and even 125 nm lipid nanoparticles (LNPs). Uptake was prevented by molecules known to inhibit eukaryotic endocytosis, suggesting this to be an energy-dependent process. Given that cell wall-deficient bacteria are considered a model for early life forms, our work provides a possible mechanism for primordial cells to acquire new genetic material or food before invention of the bacterial cell wall.

microbiology↗

bioRxiv2020

HPM Live μ for full CLEM workflow

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

biophysics↗