Samples are prepared for cryo-EM by plunge freezing in liquid ethane. This results in rapid freezing of the water into vitreous ice containing the suspended samples.
Steps of the freezing process are:
- Application of the sample to a grid.
- Blotting of the sample
- Freezing in liquid ethane
The facility is equipped with an FEI Vitrobot for automated plunge freezing. Please bring your own grids which can be purchased through several suppliers. Contact us for training on the Vitrobot!
When using the Vitrobot, the user first sets a temperature and humidity level for the upper chamber. The grid is selected and held with the Vitrobot tweezers, which are then attached to the Vitrobot and drawn up into the upper chamber. The dewar containing the liquid ethane is drawn up to the bottom of the humidified chamber. Sample is then applied to the grid via pipette and excess sample is blotted from both sides of the grid before it is rapidly plunged into the liquid ethane. The dewar and tweezers are lowered and the grid can be transferred to a storage box.
There are a number of parameters one can vary to change the behaviour of the ice on the grid:
- Absorption time of the sample before blotting
- Blot time
- Blot force
- Wait time before plunging
In practice, we find that changing blot force and blot time have the greatest effect.
See below for some helpful videos on sample preparation!