general, “tall” cells such as yeast and many eukaryotic cells may be
milled at a higher angle than unicellular bacteria, which require low
milling angles to generate enough usable area.
In order to mill at low angles, special autogrids are available
with a cutaway notch that increases accessibility at low stage tilts.
The milling notch on the autogrid should be oriented to face
towards the FIB source–the clipped grid should be rotated so that
the notch is at the top position in the shuttle. To help with orientation, we routinely mark the autogrids with colored permanent
marker to create a highly visible reference point (Fig. 2a).
2 Materials
2.1 Equipment
1. Dual beam (FIB/SEM) instrument equipped with a temperature controlled stage capable of reaching À175
C or lower and
a vacuum transfer system. This chapter is focused on preparing
specimens using the Thermo Fisher Scientific (TFS) Aquilos
system with a built-in cryo-stage and quickloader, but general
principles should be applicable across instruments.
2. Sample loading station/equipment and preparation tools compatible with the intended dual-beam instrument (Fig. 3).
3. Grid boxes.
4. Grid box opening tools.
5. Fine manipulation tweezers (e.g., Dumont #5).
6. Hot plate or hair dryer for warming/drying tools.
7. Liquid nitrogen transport dewar to hold grid boxes.
8. 4-L liquid nitrogen storage containers.
9. Personal protective equipment (PPE) including cryo-gloves
and goggles.
2.2 Consumables
1. Clean liquid nitrogen for sample preparation (NF grade; less
than 5 ppm moisture).
2. Pressurized liquid/gas nitrogen (to be regulated to 80 psi with
a capacity of 30 L/min, sufficient for 12 h use). This can be
from either an in-house line or from a separate tank, e.g., a
230 L capacity, 230 PSI tank.
3. EM grids, quantifoil type or with other film substrate (see
Note 2).
4. TFS regular autogrids or cryo-FIB-autogrids.
5. TFS c-clips, also called clip-rings or clamp rings.
Practical Approaches for Cryo-FIB Milling
57
milled at a higher angle than unicellular bacteria, which require low
milling angles to generate enough usable area.
In order to mill at low angles, special autogrids are available
with a cutaway notch that increases accessibility at low stage tilts.
The milling notch on the autogrid should be oriented to face
towards the FIB source–the clipped grid should be rotated so that
the notch is at the top position in the shuttle. To help with orientation, we routinely mark the autogrids with colored permanent
marker to create a highly visible reference point (Fig. 2a).
2 Materials
2.1 Equipment
1. Dual beam (FIB/SEM) instrument equipped with a temperature controlled stage capable of reaching À175
C or lower and
a vacuum transfer system. This chapter is focused on preparing
specimens using the Thermo Fisher Scientific (TFS) Aquilos
system with a built-in cryo-stage and quickloader, but general
principles should be applicable across instruments.
2. Sample loading station/equipment and preparation tools compatible with the intended dual-beam instrument (Fig. 3).
3. Grid boxes.
4. Grid box opening tools.
5. Fine manipulation tweezers (e.g., Dumont #5).
6. Hot plate or hair dryer for warming/drying tools.
7. Liquid nitrogen transport dewar to hold grid boxes.
8. 4-L liquid nitrogen storage containers.
9. Personal protective equipment (PPE) including cryo-gloves
and goggles.
2.2 Consumables
1. Clean liquid nitrogen for sample preparation (NF grade; less
than 5 ppm moisture).
2. Pressurized liquid/gas nitrogen (to be regulated to 80 psi with
a capacity of 30 L/min, sufficient for 12 h use). This can be
from either an in-house line or from a separate tank, e.g., a
230 L capacity, 230 PSI tank.
3. EM grids, quantifoil type or with other film substrate (see
Note 2).
4. TFS regular autogrids or cryo-FIB-autogrids.
5. TFS c-clips, also called clip-rings or clamp rings.
Practical Approaches for Cryo-FIB Milling
57