14. Quantifoil
® carbon support film is made with a plastic backing.
This should have been removed before grids are delivered but
some batches still contain the plastic which deforms during tilt
series collection and destroys the tomogram. Soaking grids in
acetone helps to remove the plastic. Chloroform and acetone
can be used instead of acetone. Acetone is very useful for
particularly troublesome batches of Quantifoil
® .
15. When placing grids into a solution, hold them perpendicular to
the surface of the solution, push them under until the grid is
fully submerged and then lay the grid down on a filter paper or
flat surface with carbon side facing up.
16. Some cell lines may not spread on holey carbon. If this is the
case, adding a thin layer of carbon will encourage them to
spread. Furthermore, certain cellular events (e.g. cell adhesion
formation) can only occur where the cell is attached to the
surface and thus a thin layer of carbon is required in order to be
able to image the event over a hole in the holey carbon
support film.
17. To add thin carbon to holey carbon EM grids, first evaporate a
thin layer of carbon onto a mica sheet using a carbon evaporator device. After a delay of at least 24 h to allow the carbon
molecules to fuse, cut the mica to the size of an EM grid, float
the carbon film onto the surface of a water drop by inserting
the mica into the water drop at a 45
angle, carbon side
up. Then push an EM grid into the water drop from the side,
near the bottom of the drop, and bring it up and out through
the carbon film. Multiple grids can be coated at once using a
device like a “Smith Grid Coating Trough”. Allow the coated
grids to dry overnight before use.
18. To help remove the carbon from the mica, gently breathe on
the carbon surface. The moisture from the breath helps dislodge the carbon from the mica surface.
19. Quantifoil
® indicates in which direction the carbon-coated
grid surface faces inside the grid box. Visually, the carboncoated side appears less shiny compared to the uncoated side.
20. To prevent removal of thin carbon film, ensure the grid enters
the cell culture media at an angle of 90
to the liquid surface.
21. If grids were coated with extracellular matrix components, this
incubation step is not necessary.
22. If cells density appears too high or too low and cells do not
spread well as a result, adjust the volume of cell suspension
accordingly.
23. It requires some experience to judge cell thickness by light
microscopy. Therefore, it might be helpful to take images on
Cryo-ET of Cellular Structures
19
® carbon support film is made with a plastic backing.
This should have been removed before grids are delivered but
some batches still contain the plastic which deforms during tilt
series collection and destroys the tomogram. Soaking grids in
acetone helps to remove the plastic. Chloroform and acetone
can be used instead of acetone. Acetone is very useful for
particularly troublesome batches of Quantifoil
® .
15. When placing grids into a solution, hold them perpendicular to
the surface of the solution, push them under until the grid is
fully submerged and then lay the grid down on a filter paper or
flat surface with carbon side facing up.
16. Some cell lines may not spread on holey carbon. If this is the
case, adding a thin layer of carbon will encourage them to
spread. Furthermore, certain cellular events (e.g. cell adhesion
formation) can only occur where the cell is attached to the
surface and thus a thin layer of carbon is required in order to be
able to image the event over a hole in the holey carbon
support film.
17. To add thin carbon to holey carbon EM grids, first evaporate a
thin layer of carbon onto a mica sheet using a carbon evaporator device. After a delay of at least 24 h to allow the carbon
molecules to fuse, cut the mica to the size of an EM grid, float
the carbon film onto the surface of a water drop by inserting
the mica into the water drop at a 45
angle, carbon side
up. Then push an EM grid into the water drop from the side,
near the bottom of the drop, and bring it up and out through
the carbon film. Multiple grids can be coated at once using a
device like a “Smith Grid Coating Trough”. Allow the coated
grids to dry overnight before use.
18. To help remove the carbon from the mica, gently breathe on
the carbon surface. The moisture from the breath helps dislodge the carbon from the mica surface.
19. Quantifoil
® indicates in which direction the carbon-coated
grid surface faces inside the grid box. Visually, the carboncoated side appears less shiny compared to the uncoated side.
20. To prevent removal of thin carbon film, ensure the grid enters
the cell culture media at an angle of 90
to the liquid surface.
21. If grids were coated with extracellular matrix components, this
incubation step is not necessary.
22. If cells density appears too high or too low and cells do not
spread well as a result, adjust the volume of cell suspension
accordingly.
23. It requires some experience to judge cell thickness by light
microscopy. Therefore, it might be helpful to take images on
Cryo-ET of Cellular Structures
19