A systematic approach of vitrification by high pressure freezing

• Published in: Methods in Microscopy Vol. 1; no. 1; pp. 31 - 48
• Main Authors: Kodera, Chie, Bret, Yann, Eyraud, Frederic, Heiligenstein, Jérôme, Belle, Martin, Heiligenstein, Xavier
• Format: Journal Article
• Language: English
• Published: 02.08.2024
• ISSN: 2942-3899; 2942-3899
• DOI: 10.1515/mim-2024-0002

Abstract 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 µ 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 (2076 bars, cooling rate above 2000 K/s). By directly measuring physical values within the HPF chamber, we evaluate the proper functioning of the equipment, contributing to the technique’s reliability. A meticulous approach was adopted for each sample type, acknowledging the uniqueness of each specimen, and associating the final sample analysis with its HPF curve, aiding in protocol optimization. Samples including human cell pellets, cell monolayer, mouse brain and liver biopsies, and Arabidopsis 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 artefacts. Overall, our work underscores the robustness and versatility of our HPM Live µ in preserving biological ultrastructure, offering valuable insights for researchers employing EM techniques in diverse biological studies.