Quick guide to use EPU for Cryo-em data collection

Load samples into the microscope. Slot 1 is reversed for the carbon cross-grid which is used for alignments. * To load, add grids into the cassette at LN2 temperatures and transfer into the NanoCab filled with LN2.

• Transfer NanoCab into the microscope, load grids and remove the NanoCab.
  Note

  When loading grids, make sure the clip side faces opposite from the direction of loading, this prevents lose-clip grids from falling inside the microscope. Check NanoCab after loading grids. If the cassette is present, add more LN2 and try to load again. If cassette is not loading, contact your facility manager for assistance.

Do inventory of grids using TEMUI when temperatures <~100Kelvin, indicated by the switch of color from red to green.

While inventory is going, go to an empty hole in the carbon-cross grid using the Preparation Tab on EPU.* Acquire image using the atlas magnification to visualize an empty hole and move to that area using right click and pressing “move here” or “move/acquire”.

Switch to data acquisition magnification using the Preparation Tab on EPU and load the preferred magnification, press SET to send it to the microscope.

In the same Tab press Measure to check the dose and change it to your preferred dose.

Move to an area that is not broken using the atlas magnification in the Preparation Tab. * Select the Autofunction Tab and select Beam alignment by beam tilt in the hole eucentric height magnification . Followed by Autofocus in autofocus magnification .

Skip this step if the microscope does not have an energy filter.

• Go to the program "Sherpa" and select the Energy Filter tab.
• Press Center (Zero loss) followed by isochromaticity measure .

Go back to the Autofunction Tab in EPU and select Autostigmate in Thon ring magnification .* Followed by Autocoma on the same magnification.

Using TEMUI, put it the objective lens and redo Autostigmate in Thon ring magnification .

Go to EPU, select the Atlas Tab and start atlas collection.

Once Atlas was collected, go to the EPU Tab and set up your session selecting the type of grid and type of acquisition.

To set up collection select squares from the atlas in square selection task .

Once squares are selected, go to hole selection task and press auto-eucentric on the first square. * After auto-eucentric, press measure holes and measure the holes on the square. (measuring the holes only has to be done once).

• Continue auto-eucentric and brush selection with the next squares.

Move to hole that will not be used for collection and select the Template definition task, and add focus, drift outside the hole and target inside the hole.

* Check template using the Template execution task .

Go to Automated Acquistion task and start collection.