cells with minimal clumping. Cell clumps will lead to poor vitrification. Cells should be concentrated or diluted to a predetermined
concentration prior to plunge-freezing.
Evaluating Grid Quality
and Milling
In both seeded and deposited cells, the ideal specimen would have
on average 1–2 cells per grid square (on a 200 mesh grid,
$6000 μm area) with minimal clumps (Fig. 5a). At the SEM, cells
should appear well hydrated, and covered in a thin layer of ice
(Fig. 5d, g). In some cases, the outline of the nucleus may be
visible. Cells that are overblotted (i.e., too dry) may appear to be
shrunken, starting to lift off from the substrate (for adherent cells),
or have craters appearing on the surface.
Flat eukaryotic cells are typically milled at moderate to high
stage tilt angles, from 15
up to 22
. FIB scanning patterns should
be positioned to mill through the bulk of the cell in order to have
sufficient support material to hold the lamella. Lamellae from cells
may extend as far as 15 μm in length and may be thicker at the rear
due to FIB beam spreading. The lamella thickness can be made
uniform through a final milling step with additional stage tilt as
described in Subheading 3.2.
3.1.2 Yeast and Tall
Eukaryotic Cells
These cells are smaller than flat cells, typically encompassing less
than 10 μm in diameter and have a roughly round shape (Fig. 5e,
h). This is likely due to the presence of a rigid cell wall that holds the
cell shape. Each lamella made in these samples will have sections of
multiple cells appearing side by side. Examples include Saccharomyces cerevisiae, Chlamydomonas. In some cases, large bacteria clusters
such as filamentous Anabaena [9] lend themselves to milling in this
manner.
Grid Preparation
These cells are typically deposited on the grid before plunging,
rather than cultured directly on the grid. Cells should be diluted
or concentrated to an empirically determined concentration prior
to plunge-freezing. In some cases (such as yeast), the cells are more
tolerant of drying due to secreted extracellular polymers or the
presence of a cell wall.
Evaluating Grid Quality
and Milling
Contrary to the case with flat cells, the ideal yeast specimen should
have clumps of 5–10 cells, with 1–2 clumps per grid square (on a
200 mesh grid, Fig. 5b). Due to the relatively small size of yeast,
these clumps are necessary to provide enough material to make
reasonably sized lamellae. At the SEM, cells should appear relatively
dry, but with enough ice to cover the cells and to bridge the gap
between neighboring cells (Fig. 5e, h). As a rule of thumb, the
clumps should look like steep hills. If there is negative curvature of
the clumps where they touch the grid surface, the cells are likely
overblotted. If needed, it is possible to mill individual yeast cells,
but with a narrow lamella about 3 μm wide instead of 10 μm [34].
60
Vinson Lam and Elizabeth Villa
concentration prior to plunge-freezing.
Evaluating Grid Quality
and Milling
In both seeded and deposited cells, the ideal specimen would have
on average 1–2 cells per grid square (on a 200 mesh grid,
$6000 μm area) with minimal clumps (Fig. 5a). At the SEM, cells
should appear well hydrated, and covered in a thin layer of ice
(Fig. 5d, g). In some cases, the outline of the nucleus may be
visible. Cells that are overblotted (i.e., too dry) may appear to be
shrunken, starting to lift off from the substrate (for adherent cells),
or have craters appearing on the surface.
Flat eukaryotic cells are typically milled at moderate to high
stage tilt angles, from 15
up to 22
. FIB scanning patterns should
be positioned to mill through the bulk of the cell in order to have
sufficient support material to hold the lamella. Lamellae from cells
may extend as far as 15 μm in length and may be thicker at the rear
due to FIB beam spreading. The lamella thickness can be made
uniform through a final milling step with additional stage tilt as
described in Subheading 3.2.
3.1.2 Yeast and Tall
Eukaryotic Cells
These cells are smaller than flat cells, typically encompassing less
than 10 μm in diameter and have a roughly round shape (Fig. 5e,
h). This is likely due to the presence of a rigid cell wall that holds the
cell shape. Each lamella made in these samples will have sections of
multiple cells appearing side by side. Examples include Saccharomyces cerevisiae, Chlamydomonas. In some cases, large bacteria clusters
such as filamentous Anabaena [9] lend themselves to milling in this
manner.
Grid Preparation
These cells are typically deposited on the grid before plunging,
rather than cultured directly on the grid. Cells should be diluted
or concentrated to an empirically determined concentration prior
to plunge-freezing. In some cases (such as yeast), the cells are more
tolerant of drying due to secreted extracellular polymers or the
presence of a cell wall.
Evaluating Grid Quality
and Milling
Contrary to the case with flat cells, the ideal yeast specimen should
have clumps of 5–10 cells, with 1–2 clumps per grid square (on a
200 mesh grid, Fig. 5b). Due to the relatively small size of yeast,
these clumps are necessary to provide enough material to make
reasonably sized lamellae. At the SEM, cells should appear relatively
dry, but with enough ice to cover the cells and to bridge the gap
between neighboring cells (Fig. 5e, h). As a rule of thumb, the
clumps should look like steep hills. If there is negative curvature of
the clumps where they touch the grid surface, the cells are likely
overblotted. If needed, it is possible to mill individual yeast cells,
but with a narrow lamella about 3 μm wide instead of 10 μm [34].
60
Vinson Lam and Elizabeth Villa