December 1st, 2nd at the University of Heidelberg, Germany
Partner: Conway Institute, University College Dublin
The Imaging Core lab of Dr Dimitri Scholz at the UCD Conway Institute is focused on the development of correlative workflows to support the virology work packages. Integration of soft X-ray tomography (SXT) into CLEM imaging workflows offers a powerful new method for 3D imaging of whole cells and tissue. SXT quantitatively and rapidly images fully hydrated, intact cells at about 50 nm resolution. Conventional TEM generates high-resolution (~5 nm) data from small regions of interest of cells. Combining these two powerful imaging techniques with data from fluorescence microscopy will provide a more informative picture.
Several potential workflows are available at UCD Conway, focusing on the correlation of three modalities:
- Light (fluorescent, including confocal and super-resolution) microscopy
- Electron (scanning as well as transmission) microscopy (SEM and TEM)
- Soft X-ray microscopy
Presently a CLEM workflow of cryo-fixation, cryo-substitution and embedding in fluorescent-friendly Lowicryl is being optimised (as depicted by the path of the yellow arrows in the figure below). Cryo-fixation is achieved either by high-pressure freezing or plunge-freezing. Both methods are well established in the Conway Imaging lab. For cryo-substitution and embedding at low temperatures, a recently purchased cryo- substitution device permits multiple CLEM workflows.
After the establishment of the CLEM workflows, we will insert cryo-SXT imaging into the workflow, followed by freeze substitution, resin embedding and conventional room temperature TEM. The benefit of this workflow will be in using the whole-cell imaging capability of SXT to identify regions of interest for high-resolution TEM imaging, thus improving the efficiency of CLEM workflows.
These workflows will be further extended to include the correlation of super-resolution STED with cryo-SXT and conventional TEM or SEM.