8. Manual or “hand” blotting from behind avoids applying pressure directly to the cells by the paper and any mechanical stress
it would induce. Cells like to hold water so it is easier to underblot than it is to over-blot. Even if parts of the grid are dry,
areas containing cells or cell processes will tend to hold enough
media to encapsulate them. The holes in the carbon support
help to allow water trapped between cells to be removed.
9. To ensure vitrification, the ethane needs to be below À160
C.
Since the Vitrobot does not have a thermal sensor in the
ethane, seeing frozen ethane forming around the inside of the
brass cup is a good visual indicator. With the outer portion of
the cryo cup filled with liquid nitrogen, and the Styrofoam
floating ring in place, it should take 5–10 min to see frost
building up. It is useful to keep a small copper rod around for
melting ethane if the whole cup freezes.
10. The grid will disassociate from the paper once most of the
media has been wicked away and there is no more surface
tension holding the grid and paper together. With enough
practice this can be observed during blotting and used as a
cue to progress with plunge freezing.
11. Make sure that any grids you load for tomography are clearly
flat as seen by eye. If there are large bends or creases in the grid,
it will be difficult to maintain eucentric height while moving
around the grid.
12. To use the K2 in live mode, the camera will need to be controlled by the Gatan Digital Micrograph Software (GMS).
Currently, there is no way to use the live mode directly through
the Tomography software. It is important not to dwell on one
spot for long periods of time during live viewing to avoid
burning through the sample. If ice is bubbling very quickly,
expand the beam or increase the spot size to minimize electron
dose.
13. The defocus value is entirely up to the user, but 6–8 μm under
focus is a good range. For more detail, collect closer to À6 μm
and for more contrast collect closer to À8 μm. For thicker
objects (>600 nm), where contrast may already be poor, it is
better to shoot near À10 μm to increase contrast. As a point of
reference, À8 μm is required to resolve the leaflets of a membrane bilayer.
14. 10 nm gold fiducials are acceptable for the magnification range
where most tomography is done. 5 and 20 nm fiducials can be
useful when collecting data at higher or lower magnifications,
respectively. As long as the fiducials can be auto-tracked by the
software, it is better to stay on the smaller side, simply to
minimize the artifacts fiducials add to the reconstruction.
While it is not covered in this protocol, Etomo can remove
the gold fiducials from the tilt-series, post-alignment.
46
Ryan K. Hylton et al.
it would induce. Cells like to hold water so it is easier to underblot than it is to over-blot. Even if parts of the grid are dry,
areas containing cells or cell processes will tend to hold enough
media to encapsulate them. The holes in the carbon support
help to allow water trapped between cells to be removed.
9. To ensure vitrification, the ethane needs to be below À160
C.
Since the Vitrobot does not have a thermal sensor in the
ethane, seeing frozen ethane forming around the inside of the
brass cup is a good visual indicator. With the outer portion of
the cryo cup filled with liquid nitrogen, and the Styrofoam
floating ring in place, it should take 5–10 min to see frost
building up. It is useful to keep a small copper rod around for
melting ethane if the whole cup freezes.
10. The grid will disassociate from the paper once most of the
media has been wicked away and there is no more surface
tension holding the grid and paper together. With enough
practice this can be observed during blotting and used as a
cue to progress with plunge freezing.
11. Make sure that any grids you load for tomography are clearly
flat as seen by eye. If there are large bends or creases in the grid,
it will be difficult to maintain eucentric height while moving
around the grid.
12. To use the K2 in live mode, the camera will need to be controlled by the Gatan Digital Micrograph Software (GMS).
Currently, there is no way to use the live mode directly through
the Tomography software. It is important not to dwell on one
spot for long periods of time during live viewing to avoid
burning through the sample. If ice is bubbling very quickly,
expand the beam or increase the spot size to minimize electron
dose.
13. The defocus value is entirely up to the user, but 6–8 μm under
focus is a good range. For more detail, collect closer to À6 μm
and for more contrast collect closer to À8 μm. For thicker
objects (>600 nm), where contrast may already be poor, it is
better to shoot near À10 μm to increase contrast. As a point of
reference, À8 μm is required to resolve the leaflets of a membrane bilayer.
14. 10 nm gold fiducials are acceptable for the magnification range
where most tomography is done. 5 and 20 nm fiducials can be
useful when collecting data at higher or lower magnifications,
respectively. As long as the fiducials can be auto-tracked by the
software, it is better to stay on the smaller side, simply to
minimize the artifacts fiducials add to the reconstruction.
While it is not covered in this protocol, Etomo can remove
the gold fiducials from the tilt-series, post-alignment.
46
Ryan K. Hylton et al.