parallel to the substrate and long enough to contain enough material of interest as detailed in Subheading 1.2. The FIB is used to mill
away material by continually rastering over the sample in a userspecified pattern to gradually ablate material. When starting a
lamella, the milling patterns are set $2 μm apart, and are gradually
brought closer together to reach the target thickness, <200 nm.
Additionally, the ion-beam current is reduced as the lamella
becomes thinner in order to afford more control over the milling
process and to minimize beam damage. The final target lamella
thickness should be informed by the biological question and any
downstream analysis (e.g., subtomogram averaging or membrane
segmentation) (see Subheading 3.3). Typical lamellae thicknesses
may range from 85 to 250 nm, and typical TEM pixel sizes may
range from 0.2 to 2 nm (see Subheading 3.3).
1.1.1 Platinum
Sputtering and GIS
Deposition
Vitrified biological samples are not conductive, and thus often
result in beam-induced charging. This charging effect can be detrimental during milling and also during TEM imaging that leads to
low quality images. A platinum sputtering system can be used to
reduce charging by depositing a thin conductive layer on the sample. The sputtering system may be built into the chamber or immediately outside the chamber as part of a cryo-stage/quickloader
installation, both enabling sputtering to be performed on cryogenic samples without excess contamination.
For cryo-FIB milling, an organo-platinum gas injection system
(GIS) is used to coat the surface of the cold grid before lamella
milling. The gas is released into the chamber close to the sample
and immediately condenses on the cold grid. When exposed to the
ion beam, the organic component is partially sublimated, leaving a
metallic/organic film behind [31]. This residual platinum is essential for protecting the leading edge of the lamella during the milling
process to prevent uncontrolled milling that will form “curtain”
artifacts [26]. Additionally, the platinum layer acts as structural
support that prevents lamella cracking during subsequent handling.
1.1.2 Stage and Shuttle
Up to two grids previously clipped into an autogrid support (see
Note 3) can be loaded into the specimen shuttle (Figs. 2a, b and
3e) for insertion into the microscope. The grids are held in place by
a metal spring flap that contacts the edge of the autogrid and
maintains thermal contact. In order to minimize atmospheric ice
contamination, the shuttle is transferred from the loading station to
the microscope under vacuum using a sealed transfer arm (Fig. 3a),
via a pumped airlock system (the quickloader, Fig. 3j). Additionally,
the shuttle has a spring-loaded “door” that covers the grids whenever the shuttle is removed from the loading station or the microscope stage to minimize atmospheric ice contamination.
54
Vinson Lam and Elizabeth Villa
away material by continually rastering over the sample in a userspecified pattern to gradually ablate material. When starting a
lamella, the milling patterns are set $2 μm apart, and are gradually
brought closer together to reach the target thickness, <200 nm.
Additionally, the ion-beam current is reduced as the lamella
becomes thinner in order to afford more control over the milling
process and to minimize beam damage. The final target lamella
thickness should be informed by the biological question and any
downstream analysis (e.g., subtomogram averaging or membrane
segmentation) (see Subheading 3.3). Typical lamellae thicknesses
may range from 85 to 250 nm, and typical TEM pixel sizes may
range from 0.2 to 2 nm (see Subheading 3.3).
1.1.1 Platinum
Sputtering and GIS
Deposition
Vitrified biological samples are not conductive, and thus often
result in beam-induced charging. This charging effect can be detrimental during milling and also during TEM imaging that leads to
low quality images. A platinum sputtering system can be used to
reduce charging by depositing a thin conductive layer on the sample. The sputtering system may be built into the chamber or immediately outside the chamber as part of a cryo-stage/quickloader
installation, both enabling sputtering to be performed on cryogenic samples without excess contamination.
For cryo-FIB milling, an organo-platinum gas injection system
(GIS) is used to coat the surface of the cold grid before lamella
milling. The gas is released into the chamber close to the sample
and immediately condenses on the cold grid. When exposed to the
ion beam, the organic component is partially sublimated, leaving a
metallic/organic film behind [31]. This residual platinum is essential for protecting the leading edge of the lamella during the milling
process to prevent uncontrolled milling that will form “curtain”
artifacts [26]. Additionally, the platinum layer acts as structural
support that prevents lamella cracking during subsequent handling.
1.1.2 Stage and Shuttle
Up to two grids previously clipped into an autogrid support (see
Note 3) can be loaded into the specimen shuttle (Figs. 2a, b and
3e) for insertion into the microscope. The grids are held in place by
a metal spring flap that contacts the edge of the autogrid and
maintains thermal contact. In order to minimize atmospheric ice
contamination, the shuttle is transferred from the loading station to
the microscope under vacuum using a sealed transfer arm (Fig. 3a),
via a pumped airlock system (the quickloader, Fig. 3j). Additionally,
the shuttle has a spring-loaded “door” that covers the grids whenever the shuttle is removed from the loading station or the microscope stage to minimize atmospheric ice contamination.
54
Vinson Lam and Elizabeth Villa