3 Methods
On-grid cryo-FIB milling is suitable for many types of biological
specimens including bacteria, yeasts, and mammalian cells. This
section describes generalized methods suitable for these three common specimen types with considerations for different approaches
required for each specimen. These instructions assume that the reader
is familiar with manipulating cryogenic samples to minimize atmospheric ice contamination and risk of sample devitrification. Always
use appropriate PPE when working with cryogenic liquids, and
always pre-cool tools in liquid nitrogen before handling the
specimens.
3.1 Sample Type
Considerations
Cryo-FIB milling is adaptable to a wide variety of cell types (Fig. 5).
Here, we offer some considerations on sample preparation and FIB
milling for three broad categories of cells. This protocol is not
applicable for bulk tissue samples due to major differences in vitrification procedures and FIB milling workflow [20, 28–30]. The
categories are not strictly limited to only those cell types described,
and we list some exceptions within the categories. In all cases, grid
preparation is critical and users should optimize the following for
their particular cell type:
1. Specimens should be thick enough to provide sufficient material to create a lamella. If the sample is too thin, the final
generated lamella will be very short and contain almost no
cellular features of interest. For these cases, milling wedges
can provide a longer electron-transparent window, albeit of
varying thickness, on which tomograms can be acquired
[21, 33].
2. Specimens should be thin enough to ensure proper vitrification
and minimize crystalline ice domains.
3. An individual milling target (whether a single cell or clump of
cells) should be large enough to support a 5–10 μm wide
lamella suitable for cryo-ET.
4. Appropriate cellular density for the cell type. Note that cell
density also affects sample thickness and vitrification.
3.1.1 Mammalian
and Flat Eukaryotic Cells
These cells are generally flat and extend several tens of microns in
diameter (Fig. 5d, g). Typically, the nucleus appears as a small hill in
the center part of the cell. Each lamella made in these samples will
be a partial section of a single cell. Examples include fibroblasts and
other cells that are cultured on substrates, and some amoebae.
Grid Preparation
Cells may be grown directly on the quantifoil grid by seeding cells
and allowing time for them to adhere. The grids should be made of
non-cytotoxic material such as gold. Additionally, these grids may
be treated with extracellular matrix such as fibronectin or poly-L58
Vinson Lam and Elizabeth Villa
On-grid cryo-FIB milling is suitable for many types of biological
specimens including bacteria, yeasts, and mammalian cells. This
section describes generalized methods suitable for these three common specimen types with considerations for different approaches
required for each specimen. These instructions assume that the reader
is familiar with manipulating cryogenic samples to minimize atmospheric ice contamination and risk of sample devitrification. Always
use appropriate PPE when working with cryogenic liquids, and
always pre-cool tools in liquid nitrogen before handling the
specimens.
3.1 Sample Type
Considerations
Cryo-FIB milling is adaptable to a wide variety of cell types (Fig. 5).
Here, we offer some considerations on sample preparation and FIB
milling for three broad categories of cells. This protocol is not
applicable for bulk tissue samples due to major differences in vitrification procedures and FIB milling workflow [20, 28–30]. The
categories are not strictly limited to only those cell types described,
and we list some exceptions within the categories. In all cases, grid
preparation is critical and users should optimize the following for
their particular cell type:
1. Specimens should be thick enough to provide sufficient material to create a lamella. If the sample is too thin, the final
generated lamella will be very short and contain almost no
cellular features of interest. For these cases, milling wedges
can provide a longer electron-transparent window, albeit of
varying thickness, on which tomograms can be acquired
[21, 33].
2. Specimens should be thin enough to ensure proper vitrification
and minimize crystalline ice domains.
3. An individual milling target (whether a single cell or clump of
cells) should be large enough to support a 5–10 μm wide
lamella suitable for cryo-ET.
4. Appropriate cellular density for the cell type. Note that cell
density also affects sample thickness and vitrification.
3.1.1 Mammalian
and Flat Eukaryotic Cells
These cells are generally flat and extend several tens of microns in
diameter (Fig. 5d, g). Typically, the nucleus appears as a small hill in
the center part of the cell. Each lamella made in these samples will
be a partial section of a single cell. Examples include fibroblasts and
other cells that are cultured on substrates, and some amoebae.
Grid Preparation
Cells may be grown directly on the quantifoil grid by seeding cells
and allowing time for them to adhere. The grids should be made of
non-cytotoxic material such as gold. Additionally, these grids may
be treated with extracellular matrix such as fibronectin or poly-L58
Vinson Lam and Elizabeth Villa