Cell Imaging Workflow

Standard cell preparation, cryo-fixation and image reconstruction procedures facilitate easy integration into cell imaging laboratory workflow

The SXT100 uses the same cell preparation, cryo-fixation and image reconstruction procedures as with cryo-transmission electron microscopy (cryo-TEM). This facilitates easy integration into cell imaging laboratory workflows and minimises the training required to operate the microscope. 

“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:

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreements No 738774, No 959776 & No 101017116