4 Notes
1. Because the grid is held at 45
and the FIB column angle
relative to the SEM column is 52
, there is a 7
difference
between the reported stage angle and the incident milling
angle. To estimate the incident milling angle, subtract 7
from the reported stage angle.
2. The EM grid should be chosen such that each grid square is
large enough to contain the widest desired lamella, but small
enough to have sufficient sample rigidity so that the lamella will
not bend and break during subsequent handling. We routinely
use 200 mesh quantifoil grids for FIB milling. The specific
pattern of holes will determine blotting speed and sample
dryness. We are routinely using R1/4 grids for mammalian
cells and R2/1 grids for bacteria. Grid material should be
gold for cells cultured directly on the grid, copper for cells
deposited immediately before plunge freezing. See Subheading
3.1 for sample type considerations.
3. The autogrid is a rigid ring support in which TEM grids are
placed and secured with a spring clip. This arrangement allows
improved handling and stability of samples. Grids may be
clipped either using the provided loading station, or in any
other suitable device that will minimize atmospheric contamination. Grids should be clipped so that the cell side is facing
towards the flat side of the autogrid. We have found clipping
grids under a very low level of nitrogen to be most efficient,
with minimal grid square breakage. Once clipped, the grid is
permanently mounted in the autogrid and is essentially impossible to remove at cryogenic temperatures without damaging
the grid.
4. On our system, the chamber base pressure at room temperature
is typically 2 Â 10
À6 mbar or better. If the base pressure is too
high, the chamber seals, airlock seals, and nitrogen lines should
be cleaned and inspected (see Subheading 3.4.2).
5. The reported stage temperature is measured from the lower
stage, which is quickly cooled by cold nitrogen gas. However,
the upper stage (where the sample sits) usually lags behind due
to its thermal mass and reduced contact area with the lower
stage. Waiting at least 30 min after full cooldown is important
to ensure that the upper stage is well below devitrification
temperature.
6. The microscope quickloader gate valve is kept locked to prevent accidental opening and venting of the chamber. The valve
will only unlock if the user has issued the “pump” command
through the button interface AND if the pressure in the
quickloader airlock is low enough. Additionally, the gate valve
Practical Approaches for Cryo-FIB Milling
77
1. Because the grid is held at 45
and the FIB column angle
relative to the SEM column is 52
, there is a 7
difference
between the reported stage angle and the incident milling
angle. To estimate the incident milling angle, subtract 7
from the reported stage angle.
2. The EM grid should be chosen such that each grid square is
large enough to contain the widest desired lamella, but small
enough to have sufficient sample rigidity so that the lamella will
not bend and break during subsequent handling. We routinely
use 200 mesh quantifoil grids for FIB milling. The specific
pattern of holes will determine blotting speed and sample
dryness. We are routinely using R1/4 grids for mammalian
cells and R2/1 grids for bacteria. Grid material should be
gold for cells cultured directly on the grid, copper for cells
deposited immediately before plunge freezing. See Subheading
3.1 for sample type considerations.
3. The autogrid is a rigid ring support in which TEM grids are
placed and secured with a spring clip. This arrangement allows
improved handling and stability of samples. Grids may be
clipped either using the provided loading station, or in any
other suitable device that will minimize atmospheric contamination. Grids should be clipped so that the cell side is facing
towards the flat side of the autogrid. We have found clipping
grids under a very low level of nitrogen to be most efficient,
with minimal grid square breakage. Once clipped, the grid is
permanently mounted in the autogrid and is essentially impossible to remove at cryogenic temperatures without damaging
the grid.
4. On our system, the chamber base pressure at room temperature
is typically 2 Â 10
À6 mbar or better. If the base pressure is too
high, the chamber seals, airlock seals, and nitrogen lines should
be cleaned and inspected (see Subheading 3.4.2).
5. The reported stage temperature is measured from the lower
stage, which is quickly cooled by cold nitrogen gas. However,
the upper stage (where the sample sits) usually lags behind due
to its thermal mass and reduced contact area with the lower
stage. Waiting at least 30 min after full cooldown is important
to ensure that the upper stage is well below devitrification
temperature.
6. The microscope quickloader gate valve is kept locked to prevent accidental opening and venting of the chamber. The valve
will only unlock if the user has issued the “pump” command
through the button interface AND if the pressure in the
quickloader airlock is low enough. Additionally, the gate valve
Practical Approaches for Cryo-FIB Milling
77