High Pressure Freezing for Correlative Light and Electron Microscopy

CryoCapCell

Vitrification since 2013

How to increase freeze-substitution and electron microscopy embedding reliabilityFreeze substitution is a crucial technique in electron microscopy, enabling the preservation of biological specimen ultrastructure. However, existing tools are primarily designed for 3 mm carriers, limiting their compatibility with larger carriers such as CryoCapsules or 6 mm carriers. In this study, we developed a novel freeze substitution tool, simply named the FS-basket, to address the challenges associated with embedding samples vitrified in larger carriers. The tool comprises a mesh with pore sizes smaller than the sample chunks, deposited below the carrier to maintain a tight seal while allowing for easy fluid exchange.

A systematic approach of vitrification by high pressure freezingThis 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.

Step 1

Culture the cells

Step 2

Fluorescence Live Cell to High Pressure Freezing (HPF)

Step 3

From vitreous to Electron Microscopy data

Step 4

Data registration

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Whether you are focusing on CLEM or on Volume EM, CryoCapCell has developped world unique tools to help you dive into biology.

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