CSB CryoEM Facility TF20 Operating Instructions SEC
05/02/16
III.
Alignments
•
The purpose of the alignment of the microscope is to ensure that the beam is parallel to the column
and that the apertures are properly centered.
•
Alignments should be done before every imaging session.
•
Only load the most recent alignments file, made by the FEI engineer. Do not save your own
alignment file. It will be deleted. The recent alignment made by FEI will only need to be touched up
and it's a good place to start.
1.
DO NOT DO ALIGNMENTS IN LOW DOSE
2. Make sure that “Low Dose” is turned off (the box will be gray)
3. Remove the Objective Aperture
: Move the small pin on the Objective aperture to the right or to where
the point is pointing to the specimen holder. This allows you to do an alignment without the objective
interfering
4. Add a spacer to the sample holder in the compustage.
*this must be done with the column valves
closed!
The spacer is typically a BIC pen cap. This spacer allows for the sample to be removed from
the beam path without breaking the seal of the sample chamber. The spacer makes the alignment
process easier.
5. Wait for the column vacuum to recover after inserting a sample. This takes ~10 minutes. You want
IPG1=6 and IGP4=1. These values can be found in “Vacuum Overview.” This is displayed on the
right side of the left computer monitor (If it is not there then it can be selected in the bottom right
corner of the left computer monitor).
6. Open column valves.
Check for the beam; If there is no beam try one of the following:
1) Move the stage (only if a sample is in the scope/beam path)
2) Lower the magnification
3) If you can’t find the beam call/find me. There may be a bigger problem
7.
Eucentric Focus:
Click on Eucentric Focus button located on the right panel, this should take you
within a few microns of true focus.
8.
Load Alignment file:
Go to the Alignments tab, click on the flapout window and under File, find the
most recent alignments file for the appropriate voltage (120 or 200kV). Click on the file, on the
Available box under the alignments file, doubleclick on the all the alignments, then click apply. *The
beam may go away but will come back when you load the FEG registry.
9.
Load FEG registry:
Go to the Search tab. Under Registries, find the most recent file. Click on the file,
then click Set. This will change your spot size to 3.
Change the spot size back to 5.
10.
Verify Settings
: Verify that you your settings are correct (magnification, spot size, beam position).
*Typically I use Mag= 135,000x, Spot size = 5, and center the beam using the trackball. ** The
intensity of the beam will need to be adjusted when changing the magnification
11. Make the beam round
. Check this at high magnification (~135kx). Condense the beam using the
intensity knob to the size of a dime or slightly smaller. At high magnification (~135kx) the beam may
look triangular. Go to Alignment tab and click on Condenser. Right click on the current setting and
copy the setting to another location (ie. copy 3 to 1). Use the Multifunction X and Y knobs on the
control panel to make the beam round. Only adjust one multifunction knob at a time. Click “none”
when finished.
