6. Acquire and save an SEM “Photo” preset image at a low
enough magnification (80Â) to see the majority of the grid.
Evaluate the grid quality and pick out appropriate target cells or
squares for lamella milling (see Subheading 3.1, Fig. 5, and
Note 8).
7. If the samples are good, discard the nitrogen from the loading
station and let the station dry. Otherwise, you may unload the
shuttle and load new specimens.
8. Move the stage to the sputtering position and activate the
platinum sputtering feature (see Note 9). When finished, click
“Recover from Sputtering” to return to the mapping position.
9. Move the stage to the platinum deposition position (GIS position), insert the GIS needle, and deposit the organo-platinum
compound for a predetermined amount of time (typically
5–10 s, see Note 10). When finished, ensure the GIS needle is
retracted and return to the mapping position.
Lamella Milling
Lamella milling can broadly be divided into three phases: initial
rough milling, thinning to about 400 nm thickness, and final
milling to target thickness (Fig. 6).
In the rough milling phase, the goal is to develop a lamella that
has been completely cleared of material throughout its length by
the bottom and top milling patterns, respectively. We commonly
refer to this as the lamella “breaking through” and is characterized
by the appearance of empty space bordering the front and back of
the lamella. This step is crucial because the lamellae may otherwise
be obscured at high tilts in the TEM. Additionally, if the lamella has
not broken through, the sample is likely too thick. A recent report
has demonstrated that milling “expansion joints” adjacent to the
lamella improves milling stability and quality [43]. In our hands,
the use of expansion joints has increased overall lamella quality and
is now a routine part of our cryo-FIB-ET workflow.
In the thinning phase, the goal is to gradually mill the lamella
without introducing curtaining artifacts (see Subheading 3.4.3).
Before reducing the milling pattern gap, each step should be
allowed to continue until no biological material appears in the
FIB milling pattern with increased software contrast. Additionally,
the lamella surface should be monitored at regular intervals using
the SEM to check for even texture.
In the final milling phase, the FIB current should be reduced to
30 pA or 10 pA. The milling patterns should be gradually moved
closer to reach the target thickness. The lamella should be monitored more frequently by SEM to check that milling is proceeding
evenly and that the GIS platinum layer is still sufficient to protect
the leading edge of the lamella. The SEM can also be used to
estimate lamella thickness by comparing the relative transparency
of the lamella at 5 kV and 3 kV [13]. During this phase, the user
Practical Approaches for Cryo-FIB Milling
67
enough magnification (80Â) to see the majority of the grid.
Evaluate the grid quality and pick out appropriate target cells or
squares for lamella milling (see Subheading 3.1, Fig. 5, and
Note 8).
7. If the samples are good, discard the nitrogen from the loading
station and let the station dry. Otherwise, you may unload the
shuttle and load new specimens.
8. Move the stage to the sputtering position and activate the
platinum sputtering feature (see Note 9). When finished, click
“Recover from Sputtering” to return to the mapping position.
9. Move the stage to the platinum deposition position (GIS position), insert the GIS needle, and deposit the organo-platinum
compound for a predetermined amount of time (typically
5–10 s, see Note 10). When finished, ensure the GIS needle is
retracted and return to the mapping position.
Lamella Milling
Lamella milling can broadly be divided into three phases: initial
rough milling, thinning to about 400 nm thickness, and final
milling to target thickness (Fig. 6).
In the rough milling phase, the goal is to develop a lamella that
has been completely cleared of material throughout its length by
the bottom and top milling patterns, respectively. We commonly
refer to this as the lamella “breaking through” and is characterized
by the appearance of empty space bordering the front and back of
the lamella. This step is crucial because the lamellae may otherwise
be obscured at high tilts in the TEM. Additionally, if the lamella has
not broken through, the sample is likely too thick. A recent report
has demonstrated that milling “expansion joints” adjacent to the
lamella improves milling stability and quality [43]. In our hands,
the use of expansion joints has increased overall lamella quality and
is now a routine part of our cryo-FIB-ET workflow.
In the thinning phase, the goal is to gradually mill the lamella
without introducing curtaining artifacts (see Subheading 3.4.3).
Before reducing the milling pattern gap, each step should be
allowed to continue until no biological material appears in the
FIB milling pattern with increased software contrast. Additionally,
the lamella surface should be monitored at regular intervals using
the SEM to check for even texture.
In the final milling phase, the FIB current should be reduced to
30 pA or 10 pA. The milling patterns should be gradually moved
closer to reach the target thickness. The lamella should be monitored more frequently by SEM to check that milling is proceeding
evenly and that the GIS platinum layer is still sufficient to protect
the leading edge of the lamella. The SEM can also be used to
estimate lamella thickness by comparing the relative transparency
of the lamella at 5 kV and 3 kV [13]. During this phase, the user
Practical Approaches for Cryo-FIB Milling
67