Bret, Y. | Search bioRxiv

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↗