L120C cryo-EM operation

Mounting a cryo holder
'''Warning: the L120C room (not the computer room) is not ventilated. Mind the oxygen level when working alone.'''

Mounting a cryo holder is essentially same as mounting a regular room temperature sample holder. The only major difference is that the CompuStage needs to be turned to -55° beforehand so that the liquid nitrogen dewar is not turned upside down in the beginning. When the CompuStage is at -55°, the dewar of the cryo holder would face roughly horizontal to the right when initially inserted to the pre-pump position.

Steps for insertion:

1. On the computer:

Start the turbo pump and wait for the 100% speed

With XYZ all at zero, turn the alpha to -55 degrees.

Retract screen and camera. Pumping time set to 180s. (60s is for room temperature work.) Check: column valve closed, turbo at 100%, cryobox retracted, stage at -55 deg.

2. Microscope room

Wear gloves to protect hand from LN2

a. Check that the shutter on the holder is closed.

b. Pull out the holder from the transfer dewar.

c. Turn the holder 90 degrees to the right. Some liquid nitrogen will flow out.

d. Gently insert the holder.

The automatic valve connecting the pre-pumping chamber to the TMP will be opened shortly after.

3. Computer room:
 * 1) Take a look at the vacuum value. The column reading should be below 15 log. There is only one column valve on the L120C protecting the gun area. Therefore if there is a vacuum crash, both the column and the projection chamber will have high readings (in red).
 * 2) Choose single tilt (ST) cryo holder on the TEM UI, click enter.

4. Microscope room:
 * 1) When the red light is off, gently turn the holder CCW to the end. The dewar opening should be facing left at 55° angle. Then lightly hold the dewar and let the vacuum to gently pull the holder into the column.

5. Computer room:
 * 1) Click the alpha button again to set the stage back to 0°. Now the dewar should be upright.

6. Microscope room:


 * Fill the cryo holder's dewar to top with Liquid nitrogen.
 * If there are other specimen on the same cryo grid box, transfer the cryo grid box to a dewar that can last for at leave an hour.
 * One can leave the transfer dewar in liquid nitrogen while checking the specimen in microscope - in case the current one is no good. However do not use a transfer dewar that has been cold for longer than 30 min - the rod housing is known to bend over time due to the coldness on one end.

'''The cryo holder should be refilled every 2-3 hours to make sure it still has enough liquid nitrogen in the dewar. Set up a timer.''' If the tip warms up, not only the specimen will be destroyed, the released water vapor will also crash the vacuum in the column.

Unmounting a cryo holder

 * 1) Always prepare the transfer stage with liquid nitrogen first, unless it is the end of the day.
 * 2) On computer, Check:
 * 3) the column valve closed,
 * 4) CompuStage reset,
 * 5) cryobox retracted,
 * 6) objective aperture removed,
 * 7) camera retracted, FluCam retracted.
 * 8) Only if the cryo-specimen is going to be recovered: turn the CompuStage alpha angle to -55°.
 * 9) Microscope room: Close the shutter on the cryo holder by pushing the knobs towards the microscope
 * 10) With thumb pushing against the front plate of the CompuStage, pull the cryo holder until stopped by the airlock. Gently turn clockwise ~120° to the end. Be prepared that the liquid nitrogen will be spilling from the dewar.
 * 11) Find a comfortable posture for your hand. With thumb pushing against the front plate of the CompuStage, pull the holder out. Insert the cryo holder to the cold transfer stage.

Preparing the cryo holder
Notice:

If there are multiple cryo holders on a same pumping station, close all needle valves on all cryo holders before any removal

The turbo pump used in the pumping stations should never be allowed to directly pump at atmospheric pressure.

Removing cryo holder from the pumping stage

 * 1) Close the valve on the cryo holder and the valve on the pumping stage.
 * 2) Stop the vacuum pump on the pumping stage. Wait for the pump to spin down.
 * 3) Unplug the cable and tubing from the cryo holder. Remove the cryo holder from the pumping stage. Use the sheath to protect the rod when transferring the holder between locations.

The needle valve on the cryo holder should be closed before the heating is turned off.

Baking the cryo holder after use

 * 1) Mount the cryo holder on the vacuum stage. The dewar opening faces down to keep its inside clean and to help the convection.
 * 2) Connect vacuum tubing, but do not open the valve yet.
 * 3) Start the vacuum pump and wait until the pressure drops to 10E-4 hPa (or Torr).
 * 4) Open the valve on the pumping stage. Observe that the pressure drops to 10E-4 hPa again.  If not check for leakage in the tubing connections
 * 5) Connect the heater cable.
 * 6) Start the heater. Set the heating to 100°C, 18 hours.
 * 7) Wait until the tip is heated to 22°C. (20-30 min)
 * 8) Open the valve on the holder's dewar.
 * 9) Leave overnight.


 * The dewar should be pumped to below 10E-5 Torr. (1 Torr ≈ 1.33 hPa. For the purpose here we can simply regard 10E-5 Torr as 10E-5 hPa).

Loading grid to the Gatan 626 cryo holder

 * 1) Place the cryo holder on the transfer dewar, check that the tip is at right position and that the shutter can be open and closed freely
 * 2) Fill the transfer dewar and the cryo holder dewar. There will be two boiling events, so do not over fill at the beginning. Do not fill liquid nitrogen over the seam inside the transfer dewar.
 * 3) If desired, plug the heater unit to the holder dewar to monitor the temperature at its tip.
 * 4) Use the clamp tool to remove the ring clamp and then use a fine-tip tweezer to remove old grid.
 * 5) When the dewars are cold, transfer cryo grid box to the transfer dewar.
 * 6) Cool a tweezer and the clamp tool with the ring clamp
 * 7) Place a specimen grid on the tip of the cryo holder
 * 8) Vertically insert the ring clamp over the specimen. There should be a click sound.
 * 9) Visually check if the ring clamp is secure and flat on the holder
 * 10) Close the shutter