Electron Tomography for cellular organelles.
Options:
- Frozen cells cultured on the holey carbon grid
- Plastic cellular sections embedded in the epoxy resin
- Platinum replicas for the membrane undercoats
Workflow:
- Wash holey carbon membrane/ gold grids with pure acetone (overnight at least)
- Natural dry
- Glow discharge the grid by the Quorum GloQube
- Culture the tissue cells on the gold grid
- Blot the access volume on the grid with the single filter paper
- Vitrification purified protein-complexes on the grid by the FEI Vitrobot
- Set the frozen grid on the Gatan cryo-transfer holder
- Insert the cryo-holder to the FEI TalosF200C
- General alignments for TEM
- Adjust auto Eucentric height & auto focus at optimised position
- Calibrate the Gatan or Fischione sample holder
- Calibrate the dynamic focus (for STEM-tomography)
- Prepare the acquisition positions (for cryo-tomography)
- Prepare step conditions before starting a tilt series
- Acquire tilt series
- Reconstruct live tilt series
- 3D-reconstruction and analysis by Inspect3D or IMOD