F20 User Guide (Starting Your Session) - SEMC TF20 User Manual

1. Make sure microscope is in good condition:
a. HT and FEG are on
Setup → "High Tension" and "Operate" are
i.
yellow
b. Column valves are closed
Setup → "Col. Valves Closed" is yellow
i.
c. EM column vacuum is stable (IGP1 = 6)
d. Microscope left in SA mag range
e. Stage neutral
Search → click "Holder" to zero out the X, Y, Z
i.
and α coordinates of the stage
f. Cold trap already filled with LN
refill every 1 - 2 hours
2. Login to leginon workstation with your LDAP account
3. Transfer cryo holder from the pumping station to transfer
station; cool down holder and insert your sample
a. See Dry Pumping Station User Guide (for cryo holders)
for detailed instructions
4. Before insertion,
a. See Cryo Holder Insertion/Retraction Guide for F20
detailed instructions
b. CHECKLIST before insertion:
1) turbo is on and ready (yellow)
2) objective aperture is out
3) flu screen of microscope is down
4) shield of cryo holder is closed
5) airlock pump time = 120s
6) reset holder
7) alpha = -60°
5. Insert cryo holder into EM.
6. After insertion, wait for IGP1 to reach <10 before opening the column valves
7. Select the camera/acquisition type needed for your session today:
a. If you plan to screen multiple cryo grids today, use the F416 camera
with Tietz TVIPS
i. This camera has faster throughput and yields higher contrast images in a single
exposure
ii. If your grid looks good, you can switch to the DE20 for data collection
b. If you plan to collect data for 2D/3D analysis, use the DE20 camera ("Cryo Data
Collection with the DE20" guide)
i. This camera has slower throughput, but can give you higher resolution details
within multiple frames of a movie

F20 User Guide (starting your session)

(can last for 16 hours);
2
F416)
Last updated 10/29/2020
for
(Cryo Screening
3