Overview

The Vitrobot is a PC-controlled semi-automated plunging device with humidity and temperature control for making vitreous ice (an amorphous, noncrystalline state) more reproducible by rapid cooling of a thin layer of aqueous sample e.g. protein/ virus in a liquid cryogen, commonly ethane, such that it is cooled about 200K in <10^-4 s. The process of blotting, plunging and vitrification is automated to ensure reproducibility.

Preparing the Vitrobot

1. Turn the Vitrobot on by using the switch on the back right hand side of the instrument.

2. Check whether there is any water remaining in the humidifier (please empty before filling, to avoid water leaking into the chamber). Attach the humidifier cable to the humidifier by aligning the red dot on the cable connector and the red dot on the locking ring on the underside of the humidifier (NOT on the main body of the humidifier) and pushing the connector gently upwards to lock in place.

3. Attach the humidifier to the bottom of the climate chamber by pushing up at the indicated position and turning it to the right (A). Fill the humidifier with 60 ml of fresh Milli-Q water using the syringe (B). After filling with water, pull back on the syringe to about 10ml (while it is still attached to the humidifier) to create a “vacuum”, which is required for it to be properly filled.

4. Open the chamber, and, using gloves (to avoid contamination of the filter paper), attach the filter paper with the white circular clipping rings to the blotting pads. The inner edge of the paper has a ridge and should be placed towards the pads, NOT the sample.

5. S et the Vitrobot to the required temperature and relative humidity, allow the chamber to acclimatise while condensing ethane and preparing grids.

6. Click Option on the screen, select the appropriate settings for your sample plunge cycle.

7. You can select “Use foot pedal” as alternative for processing, which will always follow the plunging sequence.

8. Perform a test-run of the plunging cycle to check everything is working correctly.

9. Seal the chamber by clicking on Place New Grid and then click on Continue to move the plunging rod all the way up to the chamber. This keeps the chamber in consistent temperature and humidity.

10. Ensure Vitrobot tweezers are aligned by examining them under an optical stereo microscope, making sure they are not bent and are symmetrically aligned in the following way: place on a flat surface and measure the tip to surface distance on all sides (should be equal within 0.5mm).

Preparing & Filling ethane at liquid nitrogen temperature (LN2)

Warning: Liquid ethane is highly flammable and can cause severe burns and blindness if splashed in the eye. Always wear safety glasses and lab coat when handling. The ethane filling must be done inside the fume hood.

1. Assemble the central (ethane) cup, the grid holder, spindle and floating ring inside the coolant container and cool down everything with filtered liquid nitrogen. Please use the 500ml thermos flasks with lids provided for this purpose, these stainless steel flasks insulate well and prevent the formation of ice crystals. They handle much better than the cumbersome 4l dewar flasks.

2. W
   ait until all parts of the coolant container, especially the central ethane cup, have reached liquid nitrogen temperature. This is the case after the four legs of the spindle have each experienced a boiling effect, after which the ethane cup has reached at least 185K (-88C), suitable for liquefying ethane. Make sure the level of LN2 is kept up during the filling.

3. To condense ethane into the central cup follow the separate instructions how to operate the ethane gas cylinder.

   1. P
      ut the tip of the gas outlet into the ethane brass cup on an angle and stay as close to the bottom as possible. Adjust the needle valve so that there is a steady even flow of ethane. It should take a few minutes for the cup to fill depending on the flow rate. The liquid ethane level should be touching the bottom of the spindle (blue arrow).

4. When finished filling, the regulator needs to be drained of excess ethane as indicated in the separate instructions.

5. Remove the spindle from the coolant chamber to prevent the ethane from freezing.

Reminder: the metal spindle must be removed from the container prior to vitrification!

6. Carefully transfer the coolant container with the liquid ethane to your workstation.

Standard Vitrification procedure

Vitrobot tweezers are very delicate and expensive (£800 each). Please handle them with care at all times.

Click Place New Grid to abort the vitrification process, using a foot pedal will continue in sequence!

1. Always only use dry tools to reduce ice contamination of the liquid nitrogen.

2. Anything which comes into contact with the frozen grid (e.g. the cap of the grid box, tweezers etc.) needs to be pre-cooled to prevent it from warming up.

Warning: Make sure the ethane is liquid and not fully frozen before starting the vitrification cycle or else it may bend your grids and damage the Vitrobot tweezers!!!

3. Check the humidity before each run.

4. Set your freezing parameters (see box below for recommended settings).

5. If you are using glow discharged grids or require special grid treatment, the best time for treatment is after filling the ethane cup (keeping an eye on the level of LN2, to prevent evaporation, cover plunging cup with a sheet of lint free paper), to ensure good quality grids.

6. There is no need to wear gloves for any stage of the procedure, other than installing the Vitrobot filter paper on the blotting pads. Gloves can transfer static to the grids, and cause charging later, even when using antistatic tweezers.

7. Pick up a grid with the Vitrobot tweezer. Make sure to orientate the desired side of the grid on the side where you are going to load your sample (for application through the right hand port, carbon side should be facing to the right) and also taking into account the correct mounting orientation of the tweezers (screw facing to the right!)

8. C lick Place New Grid to put the central axis of the tweezers in the correct position for mounting and mount the tweezers with the grid.

9. Click Start Process and wait for the tweezers to be lifted up into the climate chamber.

10. Place coolant container onto the lift and check the liquid nitrogen level. If it is too full, there is a danger of overflow and nitrogen burns, or if nitrogen comes into contact with the central ethane cup, the ethane will solidify.

11. Click Continue to raise the coolant container towards the climate chamber.

12. Click again Continue to proceed, the tweezers will be slightly lowered to allow the application of sample through either of the side-entry ports using a pipette.

13. Apply 3-4 µl sample to the grid and close the side-entry port. Colloidal gold solutions for tomography tracking can either be mixed with the sample before, or added separately to the sample after application to the grid.

14. Unless special incubation time is required (e.g. with extra carbon layer), click Continue to plunge the grid in the liquid ethane.

15. Once the coolant container has stopped after the plunging motion, fill the level of LN2 to cover your gridboxes making sure not to spill any LN2 inside the floating ring and/or the ethane cup.

16. U nless disabled in the options setup, a grid transfer movement will take place in the next step. Remove the tweezers from the plunging rod, taking care to neither bump the grid inside the ethane cup, nor to lift it out of the ethane, and either continue with the grid transfer step (where the coolant container will move down and forward), or transfer the grid directly to the LN2 or to the grid box (don’t leave the protective atmosphere of N2 gas!)

17. After finishing freezing your samples, seal your box(es) and transfer to a labelled falcon tube/cryo EM puck etc, pre-cooled in liquid nitrogen, for storage.

18. To prevent ice crystals forming outside the floating ring from dropping into the LN2 and contaminating it, change the floating ring frequently, taking care not to drip LN2 into the ethane cup.

19. I f the ethane starts freezing during the plunging cycle, the spindle can be placed in reverse on top of the ethane cup, and rotated gently to warm up the cup enough to avoid ethane freezing solid. Remove after the ethane has thawed (DO NOT LEAVE IN PLACE DURING PROCESSING!)

As a good starting point for vitrification…

- For 1.2/1.3 grids: 3.5­–4.5 seconds blot time and -15 blot force

- For 2/2 grids: 3.0­–3.5 seconds blot time and -15 blot force

For optimisation, adjust the blotting time to get good ice thickness, or adjust the blot force (- means more force and +means less force; at 0 the pad should be just touching the grid). Blot time has more influence on ice thickness than blot force!

Shutting down the Vitrobot and the end of the session

1. When you are finished, take the LN2 container back to the fume cupboard. Fully disassemble if it is possible to do so safely (otherwise leave assembled). Leave ethane to evaporate and pour the LN2 into the Styrofoam box next to the sink, before leaving the Vitrobot cup in the fume cupboard or the warming oven to warm up.

2. Clean the tweezers with 70% ethanol and store them in their case.

3. Exit the software by clicking exit on the screen (make sure you have removed the Vitrobot tweezers) and wait for the system to go down and the screen to go dark. After the software has shut down, make sure you turn off the Vitrobot with the switch at the back!

Warning: If the Vitrobot is not turned off with the switch at the back, the system WILL come back online after a while and stay on! Please make sure you double check!

4. Remove the blotting paper from the Vitrobot chamber and discard in the autoclave waste bin (ideally in the large bin, to avoid the Bio-Waste container from filling up fast) and leave the pair of plastic rings inside the chamber.

5. Disconnect the humidifier cable from the humidifier by pushing the locking ring on the underside of the humidifier upwards and gently disconnect the cable by pulling from the metal connector part, not the cable itself.

6. Remove the humidifier by turning right and pulling it downwards. Empty all the water out into the sink. Always leave the humidifier as empty as possible, and at an angle in the paper lined box, to facilitate further drying without preventing air circulation.

7. Perform a general clean up of the workspace and leave the Vitrobot and the room in the state which you would wish to encounter it.

8. Report any problems that may have occurred during your session to EM staff and in the logbook.

References

1. FEI Vitrobot Mk IV User manual (Doc#103261).

2. Passmore LA, Russo CJ (2016) Specimen Preparation for High-Resolution Cryo-EM Methods in Enzymology

Appendix: Vitrobot checklist

1. Always wear appropriate PPE (safety spectacles, lab coat and gloves e.g. cryo gloves when handling LN2 and liquid ethane; gloves when handling samples).

2. Turn on the Vitrobot. Check: is the humidifier dry? If so, connect it to the bottom of the climate chamber of Vitrobot and fill with 60ml MQ water using the syringe.

3. Keep the syringe attached and pull back to create slight vacuum.

4. Attach fresh filter paper to Blotting Pads using the clipping rings.

5. Go to Vitrobot User Interface:

   1. Console: set temperature and humidity to relevant temperature and humidity settings. Leave it equilibrating at least for 20 min.

   2. Options: set blot time, wait time, drain time, blot force, and blot total. Allows options for using the footpedal, turning off the humidifier during processing and skip grid transfer and autoraise ethane lift.

6. Perform a test run without tweezers to ensure everything is working and that theblotting pads are properly aligned and centred for the first blot.

7. Fill the coolant chamber with LN2. Warning: Liquid ethane is highly flammable and can cause severe burns and blindness if splashed in the eye. Always wear safety glasses and lab coat when handling. The ethane filling must be done inside the fume hood.

8. Once the coolant chamber is cooled down, slowly fill ethane into the central cup. Liquid ethane should be just slightly below the brim of the cup for optimal vitrification.

9. Remove the metal spindle. If you wait too long, the spindle will be hard to remove as the ethane will become solid and freeze the spindle into place.

10. Mount (fitting tweezers correctly!) the tweezers with a glow-discharged grid to the Vitrobot by first selecting “Place New Grid” button (central axis will be moved to loading position). Make sure the carbon side of the grid is facing to the desired side-entry port.

11. Select “Start Process” (or step on footpedal) to move the tweezers into the climate chamber.

12. Place the coolant chamber on the tray. Warning: Make sure the ethane is defrosted before start the vitrification cycle or else it may bend your grids and even the expensive Vitrobot tweezer!!! Also, check the LN2 is appropriate.

13. Select “Continue” (or step on footpedal) to raise the coolant container towards the climate chamber.

14. Select “Continue” (or step on footpedal) to lower the tweezers slightly to allow sample application through the side-entry port using a pipette. Remember to apply sample on the carbon side of the grid).

15. Select “Continue” (or step on footpedal) to activate blotting and plunge freezing together (with or without wait or drain times). Afterward, the coolant chamber and the tweezers will be lowered automatically.

16. Select “Continue” (or step on footpedal) to lift the grid from the ethane cup, transfer the grid into grid storage box.

17. When finished freezing, seal the grid box and transfer it to the storage dewar for long term storage or imaging.

18. Place the coolant container in the fume hood for evaporation of LN2 and liquid ethane.

19. Check the end of session checklist (p.8 of Vitrobot manual) to clean up and leave the Vitrobot and the room area in the state which you would wish to encounter it.

20. Report any problems that may have occurred during your session to EM staff and in the logbook.