3. Pump the nitrogen gas isolated line to reach a vacuum lower
than 5 Â 10
À2 mbar. This is important for the liquid nitrogen
cooled gas to remain cold before it reaches the stage.
4. Set the temperature of the cryo-stage and anti-contaminator to
desired number. Note that the temperature of the anticontaminator is usually 20
lower than the stage. Set gas flow
to auto. If using a self-pressured tank, make sure the pressure is
higher than 30 psi.
5. Put the cooling rod into the liquid nitrogen dewar. Wait until
the cryo-stage and anti-contaminator have reached their
desired temperatures (usually À168
C and À188
C respectively). This usually takes 30–40 min.
6. To begin the sample transfer to the SEM cryo-stage, the shuttle
and loading box should be pre-cooled with liquid nitrogen.
Grids need to be clipped into autogrids with the cell side (the
flat side) facing down before transfer to the loading box.
Fig. 2 Preparation of a lamella by focused ion beam milling. (a) SEM image (top view) of a cluster of yeast
cells. (b) FIB image (oblique side view) of the lamella after milling and polishing both the top and bottom of the
cell. (c) SEM image (top view) of the lamella at high magnification. (d) Lamella imaged by cryo-TEM
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Mohammed Kaplan et al.