Fast cryo-plunge freezing
This protocol describes sample preparation with a new cryo freezing apparatus shown on Figure 1. Once the apparatus is setup, the freezing is automatized with robotics and does not require user control.
Primarily to specimen preparation, it is required to align the instrument for handling of glass capillaries. Please notice glass capillaries of Hilgenberg design version 3.0 and higher is required. In the case of other capillaries current manual is not sufficient. Once the instrument is setup for handling of glass capillaries, it should be cooled by liquid nitrogen. For controlled cooling of the specimen, the apparatus is equipped with fry nitrogen flow control and the set of heating elements. The specimen is frozen by controlled (speed and acceleration) plunging into liquid propane. The propane is cooled via liquid nitrogen. All glass capillaries are stored in the storage packs specifically designed for this instrument.
Liquid nitrogen tank 100L and 50L
Gloves for cryo handling
Glass capillaries (Hilgenberg 3.0)
Steal holder for glass capillary
Metal plate for inserting capillary into a holder
Plastic tube with a bottle (cut out) to remove residual cold gas
Thermally isolated box for loading liquid nitrogen
Steal pack for storage
Plastic pack for storage
Thorlabs tool for transporting the packs inside freezing apparatus
Mark’s grabber to transporting pack to storage outside
Tubing to fill in propane
1. Ensure that there is enough Liquid nitrogen in 100L tank and 50L tank. Check propane. Check holders and storage capacity for capillaries.
2. Open the lid of the freezing instrument. Make visual inspection of the reservoir, propane well and heating elements to propane well. Make sure the propane in safe fill position. Install steal holder for capillaries on the left (marks on the side of it towards the corridor), install plastic storage box on the right from it.
3. Make visual inspection of the roboter below the main table (white isolated box). Ensure that there are no screws, screw drivers or anything else left to obstruct the motion.
4. Switch on freezing apparatus (main power plug below the table) and computer. Make sure that the GPAs on the table are working as well (blinking green light).
5. Startup lab view library “CURRENTPLUNGERGUI – Shortcut”. In the library startup program ”cameradriver”. Press run, wait until it shows it is in operation.(if code left over from previous day close and reload code to reinitialize!!!! Left over surprises cause trouble with startup protocole)
6. Start to home in the order from left to right: home Plunger Y, remove the safety pad, home Plunger X (if it makes sudden move to the side, home it again), home Plunger Z, home robot (If the Labviewgives error on time out press continue)
7. Go To RobotControlTab:OTHERROBOTPOSITION:PropaneMotionSafe and MoveToOtherPositions, check position of the robot with respect to the red arrow on the +corpus(!!!!IMPORTANT), home propane well, home pneumatics.
8. Make a test run with a capillary. Put capillary in the steal holder and mount the holder into specimen loader arm. Activate step 1 of specimen preparation. Adjust x and z, (y if necessary) according to the position of the capillary. Setup these new values in the automatic routine. Activate step 2. If required find the tip position.
9. Activate steps 3-5. At step 5 make sure to check the storage position for 0.
10. Repeat with the adjusted procedure another capillary and set the storage to 60. If the capillaries are stored correctly, you are ready to cool down. If not adjust offset of G and H motor and their rotation.
1. Make sure there are at least one pack and the storage box inside -> or empty if you are going to use already existing pack
2. Make sure that the robot is homed and there is space for propane well to move around.
3. Put propane well to the position as for plunging (y (absolute position) to 0, then rotation to 1800 and then back to -126000) -> click Propane to plunge position
4. Attach tubes for dry nitrogen and two tubes for gas to get out and liquid nitrogen fill in
5. Start Heating code, start gas flow by clicking on HighFlowOff then check in “on”, you should hear a click. Set the flow to be 30 (Normal is checked in). 900 for freezing down. Check that the measured flow is 900, if not check that the valve for liquid nitrogen is open, if yes click the errors up and down in the labview. The flow should be measured as set.
6. Switch on heaters to 25 degrees: TOPHEATER, Lidheater. BigExchangeHeater to 60 degrees
(changed to 40 max the output was wrong before) check the setting with the picture on the desktops
7. Fill in liquid nitrogen to cool down incoming gas first. Then start to fill freezing apparatus. Fill it until Liquid nitrogen is 1 cm below the top of the steal pack for capillaries.
8. When the gas is not coming out as strongly remove the tube and put the cap on top of propane well. Also if you do not add anymore Liquid nitrogen remove the box and replace with the cap.
9. Put propane well in Liquid nitrogen wait until it wont boil (ca. 0.9V)
10. Fill in propane.
Transfer of existing pack and storage box for reuse
1. Make sure don’t fill too much of nitrogen. Open the cover and add inside storage box with the pack
2. Close the lid, add some more liquid nitrogen on top to have more wait in the storage pack
3. Other positions: move plunge pack to storage, open robothatch, take thorlabs transfer stick and screw in the pack
4. Fix the pack, go to other positions: Move plunge to kinipick, put down the pack make sure of the right orientation and engage all 3 corners. Remove transfer. Close the hatch and move robot to propane safe position
1. Setup plunger for acceleration/deacceleration (Step 4-set AUGUSTA): 400000000 (new: 5*108), speed: 2000000 (new: 3*106)and TLE: 6 (1.13m/s).acc/deacc 4*10^8 1.78. The same acc but speed 5*10^6 is 3.125 m/s.With speed 3*10^6 is 2.5m/s Set propane under plunge position speed to 100 (no needed). Make sure the height of the propane well is set at -122000 for the plunging.
2. Make a test run with empty tube. Check grabbing, speed of plunging and storage options.
3. Run through your samples.
1. If you left box for filling liquid nitrogen, it is time to remove it.
2. Home propane well AND move to “propane plunge”
3. Move plunge puck to KINIPICK
4. Open robothutch
5. Put in Thorlabs tool, screw it in the puck and lift puck high enough for the storage box to pass by
6. Move plungepuck to storage. Put in your steal puck and unscrew the Thorlabs tool. Remove it
7. Close robothutch
8. Move robot to propane motion safe
9. Move the box to the storage. Remove the lid and move the box in the dewar.
1. Move propane to fill position Remove remaining propane
2. Switch off liquid nitrogen flow in Labview. Switch it off physically at the tank.
3. If the apparatus is still full with liquid nitrogen then leave the heater on for a few hours. Otherwise switch heaters off.
4. Home propane well
5. Put the safety cushion for the plunger
6. Switch off labview (ESC key)
7. Switch off power plug.
#3 1.6V 3.1 m/s (4*108,3*106,TL6)
#5 1.48 3.1 m/s (4*108,3*106,TL6)
#7 0.9V 3.1 m/s (4*108,3*106,TL6)
#9 0.9V 1.56m/s (4*108,2*106,TL6)
#11 0.968V, 1.5625m/s (4*108,2.5*106,TL6)
#13 1.36V, 3.1m/s, (4*108,2.5*106,TL6)
Y, x, z
42500 -487 2000
#4 IS LIGHTLY TOUCHING THE OUTERNTUBE BUT NOT INSIDE. CAN BE USED