TITLE: Quick guide to use EPU for Cryo-em data collection
AUTHORS: Marta Sanz Murillo, Amalia Villagran Suarez

[DESCRIPTION]
This quick guide provides key minimal steps for preparing the Microscope/EPU for single-particle data collection
Please note that this guide might be slightly different for each microscope and facility. 

[BEFORE_START]
Check the following before the session is started:
Microscope LN2 tank at least 20% full.
Smart EPU Software.
Carbon cross-grid inside the microscope for alignments. 




[STEPS]
SECTION: Preparation before alignments
1. 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.
 

SECTION: Preparation before alignments
2. Do inventory of grids using TEMUI when temperatures <~100Kelvin, indicated by the switch of color from red to green.
SECTION: Preparation before alignments
3. 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”. 
 
SECTION: Preparation before alignments
4. Switch to data acquisition magnification using the Preparation Tab on EPU and load the preferred magnification, press SET to send it to the microscope. 
 

SECTION: Preparation before alignments
5. In the same Tab press Measure to check the dose and change it to your preferred dose. 

 

SECTION: Alignments
7. 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. 

 

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

SECTION: Alignments
9. Using TEMUI, put it the objective lens and redo Autostigmate in Thon ring magnification. 
SECTION: Collection
10. Go to EPU, select the Atlas Tab and start atlas collection. 
SECTION: Collection
11. Once Atlas was collected, go to the EPU Tab and set up your session selecting the type of grid and type of acquisition. 

 

SECTION: Collection
12. To set up collection select squares from the atlas in square selection task.
SECTION: Collection
12.1. 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. 
SECTION: Collection
12.2. 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. 
SECTION: Collection
13. Go to Automated Acquistion task and start collection.
SECTION: Alignments
6. 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.