3. Cool down the liquid nitrogen and ethane containers with
liquid nitrogen. Once it has reached liquid nitrogen temperature remove liquid nitrogen from the ethane container and
place it in its holder.
4. Liquefy ethane in the ethane container by introducing a flow of
ethane gas with a polyethylene tubing connected to the ethane
cylinder. The gas flow can be manually adjusted by clamping
the tubing. Place the tube at the bottom of the container with a
weak gas flow until some liquid ethane forms. At that point, the
gas flow may be increased until the liquid level reaches a few
millimeters from the top of the container. Wait for the ethane
to solidify at the rim of the container, which signals that the
ethane is cold enough.
5. Adjust the liquid nitrogen level and place the container under
the plunger.
6. Fix the grid onto the plunger tweezer and transfer it into the
chamber.
7. Apply 3 μL of the sample onto the grid. In the absence of
adsorption, the sample concentration needs to be higher, in
the order of 0.3–1 mg/mL.
8. Blot the grid using the filter paper supplied by the manufacturer after having adjusted the blotting time and blotting force.
9. Plunge the grid down into liquid ethane.
10. Transfer quickly the grid into a cryo-box and store it in liquid
nitrogen.
3.2.2 Observation
and Trouble Shooting
1. Opaque grid. If your grid is not transparent to electrons, it
generally means that the ice is too thick. Blot time and force can
be increased. Make sure that reagents that increase viscosity
such as glycerol or sucrose are absent. The thickness of the
vitrified layer is of key importance to reach high resolution in
single particle cryo-EM. The thinnest is the better, and small
amounts of nonionic detergents may be added to form even
thinner layers.
2. Poor contrast. Thick ice will reduce image contrast. Some
reagents such as glucose or glycerol may affect the contrast.
Make sure that their concentration is lower than 1%.
3. Ethane contaminants. Long filaments forming aggregates are
sometimes present and originate from impurities present in the
ethane which do not evaporate in the temperature and vacuum
conditions of the microscope (Fig. 3d). Avoid solid ethane
accumulation on the EM grid which forms when transferring
the grid from the ethane to the liquid nitrogen container after
vitrification. Slow retraction of the grid from the container is
the best way to prevent ethane accumulation. Solid ethane may
Specimen Optimization for Single Particle cryo-EM
253
liquid nitrogen. Once it has reached liquid nitrogen temperature remove liquid nitrogen from the ethane container and
place it in its holder.
4. Liquefy ethane in the ethane container by introducing a flow of
ethane gas with a polyethylene tubing connected to the ethane
cylinder. The gas flow can be manually adjusted by clamping
the tubing. Place the tube at the bottom of the container with a
weak gas flow until some liquid ethane forms. At that point, the
gas flow may be increased until the liquid level reaches a few
millimeters from the top of the container. Wait for the ethane
to solidify at the rim of the container, which signals that the
ethane is cold enough.
5. Adjust the liquid nitrogen level and place the container under
the plunger.
6. Fix the grid onto the plunger tweezer and transfer it into the
chamber.
7. Apply 3 μL of the sample onto the grid. In the absence of
adsorption, the sample concentration needs to be higher, in
the order of 0.3–1 mg/mL.
8. Blot the grid using the filter paper supplied by the manufacturer after having adjusted the blotting time and blotting force.
9. Plunge the grid down into liquid ethane.
10. Transfer quickly the grid into a cryo-box and store it in liquid
nitrogen.
3.2.2 Observation
and Trouble Shooting
1. Opaque grid. If your grid is not transparent to electrons, it
generally means that the ice is too thick. Blot time and force can
be increased. Make sure that reagents that increase viscosity
such as glycerol or sucrose are absent. The thickness of the
vitrified layer is of key importance to reach high resolution in
single particle cryo-EM. The thinnest is the better, and small
amounts of nonionic detergents may be added to form even
thinner layers.
2. Poor contrast. Thick ice will reduce image contrast. Some
reagents such as glucose or glycerol may affect the contrast.
Make sure that their concentration is lower than 1%.
3. Ethane contaminants. Long filaments forming aggregates are
sometimes present and originate from impurities present in the
ethane which do not evaporate in the temperature and vacuum
conditions of the microscope (Fig. 3d). Avoid solid ethane
accumulation on the EM grid which forms when transferring
the grid from the ethane to the liquid nitrogen container after
vitrification. Slow retraction of the grid from the container is
the best way to prevent ethane accumulation. Solid ethane may
Specimen Optimization for Single Particle cryo-EM
253