CSB CryoEM Facility TF20 Operating Instructions SEC
05/02/16
22.
Select Objective aperture
: Determine which objective aperture to use (as of 3/28/16).
Position 1 = 10
Position 2 = 20
Position 3 = 40 *most commonly used for Negative stain
Position 4 = 100
23.
Diffraction:
If the objective aperture is not inserted, insert it to the appropriate setting (if the small pin
is pointing to the right/toward the specimen holder then the objective is out, if the small pin points to
the left the objective is in). Click on the Diffraction button on the right control panel and condense the
beam to a small point. Verify that the aperture is centered appropriately by observing the halo around
the small point (it should be even and continuous). Adjust as needed (usually very minimal change)
and exit diffraction mode by clicking the diffraction button.
24.
Check C2 stigmation:
Condense the beam, go through crossover and verify that the beam is round
and even. Correct if needed by going to Tune and select Condenser. (To ensure that you can get
back to where you started right click on the highlighted settings and copy them to another panel ie.
“copy 3 to 1”) Use the multifunction knobs to adjust the beam to make it round. Click
done
when
finished
25.
Check objective stigmation (done with the CCD):
Find focus (can be done using the Live View on
Digital Micrograph or by inserting the small screen and adjusting the focus until all contrast has
disappeared). Once focus has been found select reset defocus (typically R2 on the right control
board), start live view and go between 0.250.5nm underfocus (negative value). Observe the shape of
the FFT of the image. If it isn't round, go to Stigmator, select Objective and adjust with the
multifunction knobs until the FFT of the image is an even circle. Select none when finished
26. Turn on Low Dose
(If working in Low Dose mode) by clicking on the LD button, goes from grey to
yellow). This is where you will want to change/determine which magnification to use while imaging.
Most negative stain projects will use a focus magnification of 100 kx with an exposure magnification
of 62 kx80 kx (*These values are good starting points). With the large screen down, verify that the
beam is centered in all imaging modes (Search, Focus, Exposure). *Center the beam in exposure
first!!! Verify that the beam is centered and spread to the desired level in all modes and check that the
spot size is correct. Once the beam settings are correct, click through the 3 modes a few times while
centering the beam to minimize hysteresis (beam movement), when changing from one mode to the
next. *If the beam isn’t staying centered ask for help!
27. Check the liquid nitrogen in the dewar (fill every 23 hours).
28. When leaving the microscope for any reason do the following:
a Close column valves
b Stop the camera
c Retract the camera
d Put the screen down
