Yeast may be milled at moderate to high stage tilt angles, from
15
up to 22
. Milling patterns should be positioned to cut through
the center of the clump of cells. As in the case with flat cells, a final
milling step with additional stage tilt is recommended to make
uniform lamellae.
3.1.3 Bacteria
These cells are generally too small (less than 5 μm in diameter) to
mill individually. During blotting, rod-shaped bacteria (e.g., E. coli,
B. subtilis) will tend to lie down on the grid such that the long axis
of the cell is parallel to the grid surface. Examples include most
unicellular planktonic bacteria.
Grid Preparation
Bacteria are almost always deposited on the grid immediately prior
to plunging. Cells should be diluted or concentrated to an empirically determined concentration prior to plunge-freezing with the
aim to make a monolayer of cells embedded in media. The optimal
concentration is highly dependent on the specific cell shape and the
specific grid hole pattern used because the cells may slip through or
become trapped by the holes. Bacteria are generally tolerant of
drying because of the large number of closely packed cells and
secreted extracellular polymers [35]. However, the relatively large
total cell mass can lead to poor vitrification. This issue can be
alleviated through the addition of cryoprotectants such as trehalose
immediately prior to plunge-freezing.
Evaluating Grid Quality
and Milling
The ideal bacterial specimen should have a uniformly flat monolayer of cells over each grid square (Fig. 5c), with the cells packed
side by side. This arrangement will allow sufficiently long lamella
with cells throughout. At the SEM, the ice should be just enough
to cover the layer of bacteria, but without excessive hills or valleys
(Fig. 5f, i). Generally, if each bacterium is well defined, the sample is
overblotted.
Bacterial grids should be milled at low to moderate stage tilt
angles, from 11
to 15
, in order to maximize the number of cells
captured in each lamella. This will also result in a long GIS platinum
leading edge. If needed, the GIS deposition time can be decreased
slightly. A final milling step with additional stage tilt may be useful
for some bacterial samples, but is not always necessary for samples
milled at moderate stage tilts.
For FIB milling, the lamella should be targeted near the center
of the grid square for maximum tilt range at the TEM. Unlike the
case with mammalian cells and yeast, it is difficult to target one
individual bacterium for milling. Instead, the milling strategy relies
on having a near-complete grid square coverage to capture as many
cells as possible in one single lamella.
Practical Approaches for Cryo-FIB Milling
61
15
up to 22
. Milling patterns should be positioned to cut through
the center of the clump of cells. As in the case with flat cells, a final
milling step with additional stage tilt is recommended to make
uniform lamellae.
3.1.3 Bacteria
These cells are generally too small (less than 5 μm in diameter) to
mill individually. During blotting, rod-shaped bacteria (e.g., E. coli,
B. subtilis) will tend to lie down on the grid such that the long axis
of the cell is parallel to the grid surface. Examples include most
unicellular planktonic bacteria.
Grid Preparation
Bacteria are almost always deposited on the grid immediately prior
to plunging. Cells should be diluted or concentrated to an empirically determined concentration prior to plunge-freezing with the
aim to make a monolayer of cells embedded in media. The optimal
concentration is highly dependent on the specific cell shape and the
specific grid hole pattern used because the cells may slip through or
become trapped by the holes. Bacteria are generally tolerant of
drying because of the large number of closely packed cells and
secreted extracellular polymers [35]. However, the relatively large
total cell mass can lead to poor vitrification. This issue can be
alleviated through the addition of cryoprotectants such as trehalose
immediately prior to plunge-freezing.
Evaluating Grid Quality
and Milling
The ideal bacterial specimen should have a uniformly flat monolayer of cells over each grid square (Fig. 5c), with the cells packed
side by side. This arrangement will allow sufficiently long lamella
with cells throughout. At the SEM, the ice should be just enough
to cover the layer of bacteria, but without excessive hills or valleys
(Fig. 5f, i). Generally, if each bacterium is well defined, the sample is
overblotted.
Bacterial grids should be milled at low to moderate stage tilt
angles, from 11
to 15
, in order to maximize the number of cells
captured in each lamella. This will also result in a long GIS platinum
leading edge. If needed, the GIS deposition time can be decreased
slightly. A final milling step with additional stage tilt may be useful
for some bacterial samples, but is not always necessary for samples
milled at moderate stage tilts.
For FIB milling, the lamella should be targeted near the center
of the grid square for maximum tilt range at the TEM. Unlike the
case with mammalian cells and yeast, it is difficult to target one
individual bacterium for milling. Instead, the milling strategy relies
on having a near-complete grid square coverage to capture as many
cells as possible in one single lamella.
Practical Approaches for Cryo-FIB Milling
61