8. Pick up an EM grid from the 6-well plate using tweezers.
9. Transfer grid to the tweezers used by the plunge freezing
device.
10. Using a micropipette and filter paper, dropwise add and
remove a total of 20 μl concentrated 10 nm gold fiducial
marker solution to the EM grid. Do not allow the grid to dry
during this process.
11. Place a final volume of 3 μl concentrated 10 nm gold fiducial
marker solution to the grid after last blot (see Note 29).
12. Place tweezers in plunge freezing device. The side of the grid
with adhered cells should face the Teflon sheet (see Note 30,
Fig. 1c).
13. Set blotting condition (see Note 31).
14. Initiate plunge freezing routine.
15. After plunge freezing, transfer grids into cryo-grid boxes and
store them in liquid nitrogen (see Note 32). Ensure grid does
not warm up during transfer by always using pre-cooled tweezers and keeping the grid in liquid nitrogen or liquid nitrogen
atmosphere.
16. Record blotting conditions and location of grid.
17. Repeat steps 8–16 for each grid.
18. Store grids in cryo-grid boxes under liquid nitrogen conditions
until imaging (see Note 33).
3.3 Data Collection
To image cells, it is recommended to use a 300 kV cryo-TEM with
energy filter and direct electron detecting device. A phase plate can
also be useful. In this section, we focus on describing the principle
steps for recording tilt series of cells rather than microscope alignment. We presume data collection will occur at a user facility where
a specialist is available to align the microscope and assist the user.
The data collection strategy presented here is for use with the data
collection software, SerialEM (see Note 34). The basic workflow
involves obtaining a low magnification overview of the entire grid
(whole grid montage) to identify regions of interest, then a
medium magnification montage (polygon map) is recorded of
those regions to locate points of interest, and then tilt series at
the points of interest are recorded.
3.3.1 Sample Loading
and Collection of Whole
Grid Montage
1. Ensure microscope is aligned and grids are loaded.
2. Insert one grid into microscope column.
3. Set magnification to 81Â (~0.2 μm pixel size).
4. Find eucentric height: Place easily detectable feature at center
of screen. Tilt to +20
and bring feature back to center by
Cryo-ET of Cellular Structures
11
9. Transfer grid to the tweezers used by the plunge freezing
device.
10. Using a micropipette and filter paper, dropwise add and
remove a total of 20 μl concentrated 10 nm gold fiducial
marker solution to the EM grid. Do not allow the grid to dry
during this process.
11. Place a final volume of 3 μl concentrated 10 nm gold fiducial
marker solution to the grid after last blot (see Note 29).
12. Place tweezers in plunge freezing device. The side of the grid
with adhered cells should face the Teflon sheet (see Note 30,
Fig. 1c).
13. Set blotting condition (see Note 31).
14. Initiate plunge freezing routine.
15. After plunge freezing, transfer grids into cryo-grid boxes and
store them in liquid nitrogen (see Note 32). Ensure grid does
not warm up during transfer by always using pre-cooled tweezers and keeping the grid in liquid nitrogen or liquid nitrogen
atmosphere.
16. Record blotting conditions and location of grid.
17. Repeat steps 8–16 for each grid.
18. Store grids in cryo-grid boxes under liquid nitrogen conditions
until imaging (see Note 33).
3.3 Data Collection
To image cells, it is recommended to use a 300 kV cryo-TEM with
energy filter and direct electron detecting device. A phase plate can
also be useful. In this section, we focus on describing the principle
steps for recording tilt series of cells rather than microscope alignment. We presume data collection will occur at a user facility where
a specialist is available to align the microscope and assist the user.
The data collection strategy presented here is for use with the data
collection software, SerialEM (see Note 34). The basic workflow
involves obtaining a low magnification overview of the entire grid
(whole grid montage) to identify regions of interest, then a
medium magnification montage (polygon map) is recorded of
those regions to locate points of interest, and then tilt series at
the points of interest are recorded.
3.3.1 Sample Loading
and Collection of Whole
Grid Montage
1. Ensure microscope is aligned and grids are loaded.
2. Insert one grid into microscope column.
3. Set magnification to 81Â (~0.2 μm pixel size).
4. Find eucentric height: Place easily detectable feature at center
of screen. Tilt to +20
and bring feature back to center by
Cryo-ET of Cellular Structures
11