Standard cell preparation, cryo-fixation and image reconstruction procedures facilitate easy integration into cell imaging laboratory workflows
“Leica Microsystems’ automatic plunge freezer for bare grids”
Soft X-ray Tomography workflow
Step one: Sample preparation
Cells prepared by routine culture methods are grown on carbon-coated gold electron microscopy finder grids. After incubation the grids are screened in-vivo under a light microscope to check for confluency and expression of transfected proteins. Gold fiducial markers are then added to the grid to aid post-acquisition data alignment. The grid is then plunge-frozen in a cryogenically cooled fluid, typically liquid ethane, after blotting away excess fluid. The water in the sample freezes so rapidly that it does not crystallise.
“Courtesy of Linkam Scientific”
Step two: Cryo-Grid Inspection
The sample is transferred to an inverted light microscope, where individual, isolated cells can be identified for subsequent cryo-SXT. A dedicated cryo-LM stage keeps the sample in its vitrified state during grid inspection so that the grid positions of targeted cells are recorded before moving to the cryo-SXT.
Step three: Cryo-SXT
The grid is loaded into a cryo-holder that is inserted directly into the microscope via a vacuum load lock mechanism. The SXT microscope contains a visible light microscope set up to detect fluorescence, which aids in the identification of cells suitable for SXT data collection. Then a series of 2D images is acquired from different perspectives as the sample is tilted up to +/- 60 degrees.
Step four: Reconstruction & Visualisation
To date, the majority of SXT datasets have been processed with software packages that have been designed for the equivalent electron tomography data:
Using these and other available packages such as UCSF Chimera and Amira, the user can segment, colour, and cross-section the model in a variety of ways to enhance its display and presentation.
See the following link for more information on the various image analysis programs used by the SXT and EM communities: