How to Successfully Perform Live-cell CLEM

The Leica Nano Workflow

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The Leica Nano workflow provides a streamlined live-cell CLEM solution for getting insight about structural changes of cellular components over time. Besides the technical handling described in the workflow manual, this article provides additional knowledge for a successful experimental run.

For this example, the main interest was the final separation of two mitotic cells, called abscission. After cytokinesis, the two dividing cells are only connected by an intercellular bridge which needs to be resolved. Because of its size, this mechanism can’t be fully understood by live-cell imaging. Therefore, the ultrastructural analysis is needed to understand the underlying mechanism of final cell separation.

Chapter overview

  • Preparation of sapphires
  • Seeding of cells
  • Live-cell imaging
  • Freeze substitution and embedding
  • Sample retrieval and sectioning
  • Data postprocessing
  • Image correlation

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