December 1st, 2nd at the University of Heidelberg, Germany
Partner: Conway Institute, University College Dublin
Introduction:
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
Update:
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.
