Biology subjects

Heiligenstein, X.

Publications and source records attributed to Heiligenstein, X..

5 recordsLinked to original sources

bioRxiv2024

Live-HPF CLEM using the HPM Live μ: Finding back all needles in every haystack. The last frontier in CLEM

Correlative light and electron microscopy is a scientific method that encompasses several technologies and workflows. One of the pioneering workflows consisted in vitrifying by high pressure freezing a sample, shortly after live observation1. Despite its extraordinary potential, it did not turn into a routine technique for practical reasons. We redesigned the entire tool set, from the sample carrier to the high-pressure freezing machine, to standardize and democratise the technique. In our manuscript, we present all the technological developments that lead to a routine workflow for live to HPF CLEM. We demonstrate our ability to track rapidly moving endosomes live and retrieve them at the electron microscopy level, in three dimensions, with high confidence.

cell biology↗

bioRxiv2024

Vitrification by high pressure freezing of a wide variety of sample using the HPM Live μ

This study explores the efficacy and reliability of high-pressure freezing (HPF) as a sample preparation technique for electron microscopy (EM) analysis across a diverse range of biological samples. Utilizing the HPM Live {micro} technology, based on the historical hydraulic HPM010 from BalTec, we demonstrate the reliability of our industrial equipment to achieve the critical parameters necessary for vitrification. By directly measuring physical values within the HPF chamber, we ensure the proper functioning of the equipment, contributing to the techniques reliability. A meticulous approach was adopted for each sample type, acknowledging the uniqueness of each specimen, and associating final sample analysis with its HPF curve, aiding in protocol optimization. Samples including human cell pellets, cell monolayer, mouse brain and liver biopsies, C. elegans, zebrafish, and A. thaliana root and seedlings were processed for EM analysis following HPF. The ultrastructure of each sample type was rigorously examined, revealing homogeneous preservation and minimal ice nucleation artifacts. Challenges such as plant leaf vitrification were addressed, highlighting the importance of methodological adaptation. Overall, our findings underscore the robustness and versatility of our HPM Live {micro} in preserving biological ultrastructure, offering valuable insights for researchers employing EM techniques in diverse biological studies.

cell biology↗

bioRxiv2021

Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments

Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and, second, how their catabolism is intracellularly processed. Using scanning-transmission electron microscopy, electron tomography and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.

cell biology↗

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↗

bioRxiv2019

Dynamic-ultrastructural cell volume (3D) correlative microscopy facilitated by intracellular fluorescent nanodiamonds as multi-modal probes

Three-dimensional correlative light and electron microscopy (3D CLEM) are attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. In order to perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FNDs is biocompatible and easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the 3D CLEM method by registering multi-modal datasets.

cell biology↗