Using the Gatan CP3 for cryo-EM sample preparation

What does the CP3 do?

The Gatan CP3 is a plunge freezing tool used to vitrify cryo-EM samples.
The picture below shows the basic components within the Gatan CP3:

What does the CP3 look like?

The instrument has two main components:

The Main Unit

The LN/ethane holder

The Main unit has a plunge rod, timer controls, and pneumatics air system that can power the parts and control the blotting. The LN/ethane holder is the section that is detachable and is where the actual freezing process happens. There is a more detailed description of each component available below.

How does the CP3 work?

There are essentially three main steps in the process

  1. Attach a dangling tweezer with a grid into the humidity chamber and apply sample to the grid
  2. Adjust the timer so that the blotters can wick away excess liquid from the grid and control ice thickness — important cryo-EM data quality
  3. Press the start button to blot the grid and immeidately plunge the sample into liquid ethane

The grid is frozen by quickly dropping it into a small canister with liquid ethane at temperatures lower than -180C. This will vitrify the sample and prevent water crystals from forming. Vitreous ice means the water is frozen so quickly that water molecules do not have time to form crystals. You need vitreous ice so you can “see through” the grid when you are checking on the electron microscope.

Components within the Main Unit:

Humidity chamber

Blotters

Hygrometer

Sponge

Plunger

Humidity chamber. This area is designated for holding the suspended tweezers, and, as the name implies, it keeps the sample in a humid chamber to prevent the small volumes of liquid on the grid from evaporating. When working with cryo-EM sample preparation, scientists are working on the order of ul — or thousands of a milliliter.

Blotters.The blotters are what physically touch the grid when it is suspended from the tweezers in the humidity chamber. The time they will touch your sample is controlled on the order of sec and millisec using the blotting timer gauge on the top portion of the Main Unit–it has a digital display that can show the time. There are two, removable blotters mounted on either side of the CP3. The blotters are meant to be re-loaded with new blotting paper, such as Whatman 1, for every session. The blotters also have 1 side mark which indicated usage. In practice, only 1/4th of the blotting paper is used per blot, so instead of reloading blotting paper for every time you blot, you can rotate the blotters–using the white line as a guide–and blot 4X to maximize blotting paper utility.

Hygrometer. When working with such small amounts of liquid in cryo-EM sample preparation–microliters–evaporation is a big risk. To circumvent this issue, the CP3 uses a humidity chamber where your grid+sample is suspended in. The hygrometer is a metal rod located within the humidity chamber than can read out the humidity of the chamber in percent values. A standard operating humidity for sample preparation in the CP3 is anywhere from 80-100% humidity.

Sponge. The sponge is a long, rectangular, removable element in the CP3 that is soaked and inserted into the humidity chamber before operating the machine. It is a constant source of humidity that can help prevent sample evaporation. Inserting a wet sponge in the CP3 is among the first steps in grid freezing.

Plunger. The plunger is where the sample and tweezers are suspended from. There are custom tweezers with a hole on the top that are used specifically for suspending them in the plunge rod. The plunge rod is a metal piece with a handle on top that goes through the humidity chamber. Also, there is a blue button on the plunger. This is used to indicate the direction in which you need to insert the tweezers. Blue button facing you = tweezers + grid surface facing you. This piece is essential in the freezing process.

Components within the workstation:

Ethane cup

Liquid nitrogen holder

Liquid nitrogen filling cup

Cassette holder

Ethane cup Ethane is the key element in cryoEM sample preparation. Ethane, in liquid state, is colder than -180 degrees Celsius. At this temperature, water will freeze so quickly, it cannot form crystalline ice—it forms vitreous ice. Vitreous ice is needed for data collection in cryoEM. In this reservoir, ethane, in gas form, is dispensed into the liquid nitrogen pre-cooled, empty, ethane cup. When cold enough, as the ethane gas is dispensed, it will turn into a liquid. Ethane is very flammable, so it should be handled with caution

Liquid nitrogen holder.The area around the ethane cup is meant to hold liquid nitrogen. The whole workstation is insulted to keep the entire unit cold. There is a metal mesh at the bottom that will keep grids from floating to the bottom. The first stage in using the CP3 is to fill this unit ¾-full with liquid nitrogen. The liquid nitrogen will evaporate during the cool-down process, so intermittent refilling is needed

Liquid nitrogen filling cup. Air moisture contamination in the workstation is an issue in grid preparation. If you freeze grids and moisture from the air contaminates your sample, you will see thick chunks of crystalline ice on your grid on the scope—hindering data quality. Therefore, to avoid this, the main opening to the workstation can temporarily covered, and liquid nitrogen can be filled through the liquid nitrogen filling cup on the side of the workstation.

Cassette holder. Grid cassette can be held in place in the grid cassette holder at the bottom of the workstation. Grid cassettes can store 4 grids. It’s critical to clearly label cassette holders before use to keep track of cryoEM samples. At the conclusion of grid freezing on the CP3, grid cassettes are stored in liquid nitrogen tanks for long term storage.

Other components not included in the CP3:

Air tank

Blotter accessories

Blotting paper

Ethane tank

Gloves

Grids

Pipettes and tips

Plunging tweezers

Power source

Sample

Tweezers