Collaborative development and dissemination of workflows and techniques for using Correlative Light, Electron and X-ray Microscopy to progress research into the understanding and treatment of diseases

Project Information

CLEXM

Grant agreement ID: 101120151

DOI

10.3030/101120151

EC signature date 9 June 2023

Start date 1 September 2023

End date 31 August 2027

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 2 480 594,40

Coordinated by

UNIVERSITY COLLEGE DUBLIN, NATIONAL UNIVERSITY OF IRELAND, DUBLIN
Ireland

Periodic Reporting for period 1 - CLEXM (Collaborative development and dissemination of workflows and techniques for using Correlative Light, Electron and X-ray Microscopy to progress research into the understanding and treatment of diseases)

Reporting period: 2023-09-01 to 2025-08-31

Summary of the context and overall objectives of the project

The CLEXM project responds to a growing need for advanced imaging workflows that capture biological processes at multiple scales with accuracy and reproducibility. Traditional approaches such as Correlative Light and Electron Microscopy (CLEM) provide valuable insights but face bottlenecks including artefacts, limited molecular context, and challenging sample preparation. Europe has prioritised innovation in life sciences and biomedical technologies, and CLEXM contributes directly to this agenda by enabling researchers to image cells and tissues in near-native states while combining structural and molecular detail.
CLEXM pursues five objectives: (RO1) optimise reproducible cryo-sample preparation across imaging modalities; (RO2) demonstrate the benefits of multimodal workflows through various use case studies; (RO3) develop advanced computational tools for image correlation and analysis; (RO4) provide workshops and training events for CLEXM doctoral candidates; and (RO5) disseminate findings widely. Together, these objectives connect technical progress to societal impact.
The project is already delivering. Workflows have revealed new insights into virus–host interactions, nanoparticle trafficking, and the molecular basis of hearing loss. Industrial partner SiriusXT advances impact further by pioneering the first benchtop soft X-ray microscope, ensuring knowledge transfer to real-world applications. Open access publishing, preprints, and outreach activities ensure early visibility, while sustainability is promoted through adoption and adherence to the MSCA Green Charter. By equipping young scientists with cutting-edge skills, CLEXM contributes not only to imaging science but also to Europe’s long-term goals of excellence, sustainability, and innovation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project has made major progress in sample preparation, refining plunge-freezing and high-pressure freezing to preserve delicate structures across diverse models, from cultured mammalian cells to C. elegans and mice cochlea samples. These improvements underpin compatibility with multiple imaging modalities.
Building on this, multimodal imaging pipelines have been established that integrate soft X-ray tomography (SXT) with CLEM. Figures 1–3 illustrate this multiscale approach on selected application cases, spanning single cells to whole organisms. Imaging across scales links molecular markers with ultrastructural context and provides a versatile platform for case studies in infection biology, nanomedicine, and cilia disease mechanisms.
On the computational side, innovative strategies have been introduced for registration and correlation. A binary-mask approach using organelles such as lipid droplets improved accuracy, while hybrid alignment methods combined principal axis fitting with coherent point drift to overcome rotational mismatches. Deep learning pipelines were also explored through data simulation. Although classical approaches remain more precise, these developments lay the foundation for introducing automation and machine learning into multimodal image analysis, a critical step for reducing manual workload and bias.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

CLEXM has demonstrated that cryo-SXT can be integrated with fluorescence and electron microscopy to produce 3D, multiscale views of biological systems. This strengthens Europe’s leadership in correlative imaging, a rapidly expanding field that is attracting international interest. The workflows enable high-resolution imaging of cells in their near-native state, revealing new insights into processes such as West Nile virus infection and nanoparticle trafficking, while also providing structural context in more complex models, including cochlear tissue and cilia in C. elegans.
For broader uptake, several needs have been identified. Cryo-sample preparation remains a global challenge across imaging communities; minor inconsistencies can compromise entire experiments. By refining reproducible protocols, CLEXM directly contributes to overcoming this barrier, benefiting both the consortium and the wider structural biology field. Further investment in automation and AI-driven analysis is required to accelerate data interpretation. The SXT100, as the first of its kind benchtop soft X-ray microscope, complements synchrotron and cryoEM facilities by providing additional capacity and flexibility, helping to alleviate scheduling pressure and ensuring more equitable access to cryo-SXT within the consortium and beyond.
With these measures in place, CLEXM will continue to bridge scientific innovation, supporting applications in pharma, nanomedicine, and structural biology.

SXT shows structural changes in liver cells before and after antiviral treatment.

FM and SXT show nanoparticle uptake and interactions with internal organelles.

FM and EM overlay reveal lipid droplets and gut granules in worm tissue.