the light microscope and compare with cell appearance by
cryo-TEM.
24. The gold fiducial markers are required for later tilt series alignment as they act as fiducial markers. Alternatively, “Fiducialless
alignment” (without gold tracer) can be performed in IMOD.
However, we highly recommend the use of gold tracer as
fiducial marker.
25. For a one-sided blotting device, e.g. the Leica GP2, the Teflon
sheet is not required.
26. Always ensure liquid nitrogen in Styrofoam dewar is at an
adequate level. This should be just above the metal bar that
holds the grid boxes.
27. Splashes from liquid ethane can cause cryo-burns instantaneously. Proper personal protection equipment should be
worn including eye protection, lab coat, and gloves. We like
using thin cotton gloves with plastic finger tips (electrician
gloves) covered with nitrile gloves rather than the thick blue
cryo-protection gloves. This maintains flexibility and dexterity
while protecting against splashes.
28. When liquifying ethane, aim the nozzle against a cryogencooled metal surface. As the ethane starts to liquify, keep the
nozzle just below the surface of the liquid ethane. When
removing the nozzle from the liquid ethane, turn off gas to
prevent splashing as the nozzle is removed. Take extra care
when placing the nozzle down as it often contains liquid
ethane.
29. If “Fiducialless alignment” is preferred, wash grid with cell
culture media or PBS (with calcium and magnesium) and
place 3 μl of the respective liquid on grid after last blot.
30. For single-sided blotting devices, ensure that cells face away
from pad with filter paper. If cells face blotting paper, they can
rupture or be pulled off the grid.
31. Blotting conditions need to be determined empirically for each
sample and each device. Suggested starting conditions for the
Vitrobot Mark IV are: 2.5–4 s with blot force 10 and 0–1 s
drain time after blotting before plunge freezing.
32. To prevent sample contamination by breathing on sample or
liquid nitrogen container, consider wearing a mask or face
shield.
33. For work on Titan Krios, grids need to be clipped in autogrid
holders using c-clips and base rings. These consumables can be
obtained from Thermo Fisher Scientific.
34. Helpful tutorials for general usage of SerialEM can be found
here: https://www.youtube.com/user/BL3DEMC.
20
Daniel Serwas and Karen M. Davies
cryo-TEM.
24. The gold fiducial markers are required for later tilt series alignment as they act as fiducial markers. Alternatively, “Fiducialless
alignment” (without gold tracer) can be performed in IMOD.
However, we highly recommend the use of gold tracer as
fiducial marker.
25. For a one-sided blotting device, e.g. the Leica GP2, the Teflon
sheet is not required.
26. Always ensure liquid nitrogen in Styrofoam dewar is at an
adequate level. This should be just above the metal bar that
holds the grid boxes.
27. Splashes from liquid ethane can cause cryo-burns instantaneously. Proper personal protection equipment should be
worn including eye protection, lab coat, and gloves. We like
using thin cotton gloves with plastic finger tips (electrician
gloves) covered with nitrile gloves rather than the thick blue
cryo-protection gloves. This maintains flexibility and dexterity
while protecting against splashes.
28. When liquifying ethane, aim the nozzle against a cryogencooled metal surface. As the ethane starts to liquify, keep the
nozzle just below the surface of the liquid ethane. When
removing the nozzle from the liquid ethane, turn off gas to
prevent splashing as the nozzle is removed. Take extra care
when placing the nozzle down as it often contains liquid
ethane.
29. If “Fiducialless alignment” is preferred, wash grid with cell
culture media or PBS (with calcium and magnesium) and
place 3 μl of the respective liquid on grid after last blot.
30. For single-sided blotting devices, ensure that cells face away
from pad with filter paper. If cells face blotting paper, they can
rupture or be pulled off the grid.
31. Blotting conditions need to be determined empirically for each
sample and each device. Suggested starting conditions for the
Vitrobot Mark IV are: 2.5–4 s with blot force 10 and 0–1 s
drain time after blotting before plunge freezing.
32. To prevent sample contamination by breathing on sample or
liquid nitrogen container, consider wearing a mask or face
shield.
33. For work on Titan Krios, grids need to be clipped in autogrid
holders using c-clips and base rings. These consumables can be
obtained from Thermo Fisher Scientific.
34. Helpful tutorials for general usage of SerialEM can be found
here: https://www.youtube.com/user/BL3DEMC.
20
Daniel Serwas and Karen M. Davies