On 14 and 15 October 2024, a workshop dedicated to the Correlative Light and Electron Microscope (CryoCLEM) was organised at the IECB and the BIC by the CLEM working group of France BioImaging (FBI): Noémie PIED (Research Engineer, BIC), Monica FERNANDEZ-MONREAL (Research Engineer, BIC) and Robin ANGER (Research Engineer, IECB). For two days, five participants from international institutions received an in-depth introduction to this innovative technique, which included theoretical and practical sessions.

What is the Cryo CLEM technique ?

CryoCLEM is an advanced technique that combines electron and fluorescence cryo-microscopy. This method enables the observation of cryofixed biological samples, preserved as close as possible to their native state.
This correlative approach draws on the advantages of both techniques, offering rich and comprehensive results :

• Fluorescence microscopy makes it possible to visualise specific structures or proteins using fluorescent markers, facilitating their identification and localisation in the sample.
• Electron microscopy provides high-resolution images, enabling the structure of cells and components to be observed in exceptional detail.

By combining these two methods, cryoCLEM offers a unique and comprehensive view of cellular organisation and molecular interactions in their natural context. Today, this technique is an essential tool for advanced research in cell biology and the study of complex biological processes.

What were the aims of this workshop ?

The aim of the workshop was to present the complete cryoCLEM workflow, covering every crucial stage from sample preparation to image acquisition and processing, providing detailed protocols and personalised advice to participants. The aim was that, by the end of the workshop, each participant should have a thorough understanding of the methodology and be able to integrate it into their own research project.

What was the programme for this workshop ?

To achieve these objectives, the workshop programme was structured in several stages over two days:

• Theoretical approach and practical session on the cryo-fluorescence at the BIC

During the first morning, a series of interactive presentations given by different experts illustrated the application of cryoCLEM in various contexts in cell and structural biology. The theoretical aspects of the technique, its advantages and its limitations were discussed during the various presentations.

Anna Sartori Rupp from the Institut Pasteur presenting her projects using cryoCLEM

In the afternoon, participants were introduced to sample cryofixation, using cryo-plungers (such as ThermoFischer's Vitrobot and Leica's EM GP2) to freeze samples on grids. They then learned how to clip the grids to prepare them for observation. The session continued with image acquisition on the Leica Cryo-CLEM fluorescence cryo-microscope, enabling participants to make their first observations of biological structures in a cryofixed state.

• Demonstration of cryo-electron microscopy and image reconstruction at the IECB

The morning began with a demonstration and practical session in which participants learned how to insert the grids prepared the previous day into a cryo-electron microscope. The organisers explained the correlation principle used to identify specific areas of interest on the grids, and then demonstrated the configuration of a tomography session. Participants were able to watch the live acquisition of cryo-tomograms using SerialEM software, giving them a practical and detailed view of this critical stage.

Acquisition of cryo-tomograms on the IECB's Talos Arctica

The afternoon was devoted to a demonstration of the processing and reconstruction of cryo-tomograms using several specialist software packages (AREtomo and IMOD). This in-depth presentation of reconstruction tools and techniques gave participants a better understanding of the processes involved in analysing and displaying cryo-microscopy data.

Will this workshop be scheduled for 2025?

The excellent feedback we received from participants showed that there was a real interest in the workshop on cryoCLEM, which met researchers' expectations while identifying areas for improvement for future sessions. We are therefore planning to hold the workshop again, but probably at a later date than 2025, in order to refine and enrich the content based on the feedback received. This period of preparation will enable us to offer even more comprehensive and optimised training in future editions.