Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

The manual plunging method described here is a cost-effective, reliable method for preparing samples for transmission electron microscopy. In this video, we will demonstrate how to set up the manual plunger, prepare cryogen, and perform manual blotting and plunging of EM grids. Prepare the Manual Plunging Environment.

This procedure should be performed in a cold room maintained at 40 degrees Celsius with humidity of greater than 95%We recommend using the materials shown here and as described in the online protocol for manual blotting and plunging. Prepare the cryogen Dewar and Manual Plunger. To assemble the cryogen Dewar, first install the platform base at the bottom of the blue styrofoam Dewar, ensuring that it is flat and level.

Then place the ethane vessel holder on top of the platform base and insert the brass ethane vessel. Finally, situate the spinning grid storage platform. Next, locate the plunging Dewar to the base of the manual plunger.

Center the ethane vessel below the plunging arm and secure the plunging Dewar to the base. To set the manual plunging height, first attach fresh tape to the base of the plunging arm. Next, fasten a pair of clamping tweezers by securely wrapping the tape around the plunging arm and the tweezer base.

Ensure the tweezers are firmly attached by tapping them lightly. Hold the plunging arm and slowly lower the tweezers into the brass ethane vessel by depressing the foot pedal. Adjust the location of the plunging Dewar so that the tweezers locate to the middle of the brass ethane vessel.

The tips of the tweezers should not contact the size or bottom of the brass ethane vessel to avoid damage to the tweezers and manual plunger. Once the position is set, reset the manual plunging arm. Prepare the Cryogen.

Cryogen preparation should be performed in a well ventilated room and proper personal protective equipment should be worn at all times. To begin, poor liquid nitrogen directly into the plunging Dewar, stopping when the liquid nitrogen level reaches the top of the brass ethane vessel. The system should be cold enough to proceed after the liquid nitrogen stops bubbling violently.

This will take approximately five minutes. Ethane comes as a compressed gas and needs to be condensed. To set the gas flow for optimal ethane condensing, insert the tip of the ethane gas line into a 250 mil beaker of water and observe the bubbling.

The gas flow should produce a steady stream of bubbles that does not violently disrupt the surface of the water. To begin condensing ethane, place the tip of the ethane gas line at the bottom of the brass ethane vessel. Slowly stir the metal tip and begin liquefying the ethane.

As soon as the ethane begins to cool, it'll make an audible noise. Fill the brass ethane vessel to three quarters full of liquid ethane. Top off the plunging Dewar with liquid nitrogen, ensuring that it touches the brass ethane vessel but does not spill into it.

The addition of liquid nitrogen is necessary for uniform ethane freezing. Place the foam lid on the blue plunging Dewar, and allow the ethane to fully solidify. Restart the ethane gas flow and carefully place the tip of the gas line into the solid ethane.

It is important to place the tip vertically into the vessel and dispense ethane at the bottom. Slowly stir the metal tip to melt the ethane and bring the level of liquid ethane up to the top of the vessel. Close the lid for approximately one minute and allow the ethane to solidify around the edges of the brass vessel until two to three millimeters of solid ethane are visible.

The ethane is now at a temperature ideal for freezing biological samples. Attach the plunging Dewar carefully to the base of the manual plunger. Fasten a pair of clamping tweezers by securely wrapping the tape around the plunging arm and the tweezer base.

Hold the plunging arm and slowly lower the tweezers into the liquid ethane by depressing the foot pedal. Adjust the location of the plunging Dewar so that the tweezers locate to the middle of the liquid ethane, avoiding contact with the solid ethane ring. Prepare Plunging Materials and Accessories.

Prepare the blotting paper by cutting each circular filter paper into approximately one centimeter wide strips. Avoid touching the center of the blotting paper and discard the smaller end pieces. It is important to ensure that the blotting paper strips are dry, clean, and free of contaminants.

Prior to plunging, place the grid boxes into the plunging Dewar into the proper slots. Ensure the grid covers our free to rotate. Prepare CryoEM Specimens by Plunge Freezing.

Due to the diversity of grid and grid preparations, follow recommended protocols for rendering the grids hydrophilic. To properly handle the grids, use clean, dry clamping tweezers to pick up the hydrophilic grids at the outer ring. Slide the plastic clamp to secure the grid and tap the tweezers gently to ensure that the grid is properly clamped.

Apply three microliters of sample to the front of the grid held in the clamping tweezers. Attach the tweezers firmly to the manual plunging arm by wrapping the tape around the tweezer base. The sample is now ready to be blotted and plunged frozen.

Hold each end of a clean strip of blotting paper between your index fingers and thumbs. Rest your hands on the edge of the plunging Dewar to establish a stable position. The blotting paper should be approximately one centimeter from the surface of the grid.

Bend the blotting paper until the center contacts the grid. It is important that the blotting paper is parallel to the grid surface to allow for even contact and to prevent grid damage. When the mobile liquid front stops spreading, begin counting.

Blot the grid for five seconds. Snap your fingers apart, straining the blotting paper and rapidly removing it from the surface of the grid. Simultaneously, press the foot pedal to release the manual plunging arm, plunging the tweezers holding the grid into the liquid ethane.

Keeping the freshly frozen grid in the liquid ethane, carefully detach the tweezers from the manual plunging arm. Carefully un-clamp the tweezers so the grid is removable. Move the grid into the liquid nitrogen reservoir below the brass ethane vessel with one swift motion from the ethane to the liquid nitrogen.

Place the grid in the grid box. Representative Results. From this video, researchers should be able to prepare CryoEM grids, such as this, with many squares containing thin films of vitreous ice for high resolution imaging.

The sample concentration and blotting time can be adjusted to optimize particle density. Here, we will demonstrate the full workflow for freezing CryoEM samples using the blot and plunge method. Use a clean clamping tweezer to pick up a fresh grid and secure the grid in place.

Apply three microliters of sample to the hydrophilic side of the grid and secure tweezers to the manual plunging arm by wrapping the tape around the tweezer handle. Position the blotting paper parallel to the grid surface and gently bend the blotting paper toward the grid to initiate blotting. After the desired amount of time, move the blotting paper in a snapping motion away from the grid surface while depressing the foot pedal to release the plunging arm, plunging the grid into the liquid ethane.

Carefully unwind the tape from around the tweezers and the manual plunging arm. With one swift motion, quickly transfer the grid from the ethane vessel into the liquid nitrogen reservoir. Place the grid in the grid storage box and wrap the tweezers in a Kim Wipe to dry;repeat.