Vitrobot MarkIV notes



Video - Mounting the Vitrobot humidifier

          - Filling the Vitrobot humidifier

          - The Vitrobot user interface and options


Setting Conditions

Main user interface

Set the proper temperature and humidity here.

Make sure that the humidity control is on since the default for startup seems to be off.


Console interface

Set blot time, force, wait time, blot total (remember to set it to “1”. Otherwise, it won’t blot);

Click the checkbox of “humidifier off during process” and “skip grid transfer”


MS notes – usually blot force 3 and blot time 8 sec work for most of single particle samples with glow discharged Quantifoil grid, and blot 0 and blot time 12 sec for single particle samples with glow discharged C-flat grid.


Equilibration and set up

• Let chamber and Vitrobot equilibrate to the set temperature and Humidity for approximately 30 minutes.
• Replace the blot paper before use.
  Video - Replacing Vitrobot blot pads

Cooling the cryogen

• Fill the reservoir and the brass cryogen cup with liquid nitrogen


• Filling the ethane cup


The reading of psi will drop
from ~5 to 0

Close the black valve

Please close ALL the
valves, vent the ethane
gas and sign on the log



Video - Plunge freezing with the Vitrobot
Attach a grid to Vitrobot tweezer

Tips – Bring the black clamp of the tweezers to the 1st resist, to lock the grid

Please be gentle.


Click “Place new grid”,
and the metal rod will
extend a few cm out.

Attach the tweezer to
the metal Rod

Double check the
tweezer is aligned in the
center of the metal rod


Place ethane container
to lift the cryogen

Start process > the
tweezer will move lower
in the chamber

Apply the sample >
“Continue” > tweezer
retracted and blotting &
freezing starts



Detach the tweezers
and put it back in the
environmental chamber

• Exit the program
• Empty the reservoir of the humidifier and lay it on its side
• Power off Vitrobot

Sign the log books and store your samples


• Note - In order to disengage the plug, you must press up adjacent to where the plug is connected under
the humidifier.


- Notes by Ming Sun