T12 Low Dose Setup Protocol
1. Click Low Dose from the Low Dose tab in the Tecnai User Interface (will turn yellow when Low Dose is
2. Set up Search mode:
a. Click the Search button.
b. Set the magnification to less than 10% of your exposure
magnification (~3000-5000x is usually sufficient for
cryoEM with holey carbon films).
c. Set the illumination conditions (condenser aperture, spot size,
beam diameter) to make the beam as dim as possible yet still
allow the user to judge the quality of the sample on the main
d. Example conditions: 150m condenser aperture, 4200x, spot
3. Set up Exposure mode:
a. Click the Exposure button.
b. Set the magnification to the desired final magnification.
c. Center and spread the beam until it reaches the edge of the main viewing screen (this diameter is
i. The beam diameter
should not be
throughout the rest
of the low dose
they should be
make sure nothing
has moved and re-
adjust if necessary.
d. Measure the electron dose
in the Exposure mode.
i. Open the flapout
box and click on
the Options tab.
ii. Make sure that
165mm is in the
beam diameter box
at the bottom of the
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iii. Click the Measure button.
iv. The readout shows the dose in e /nm /s. Move the decimal two places to the left to get
e /Å /sec.
v. If the dose needs to be changed (increased or decreased), the following can be changed:
1. Condenser aperture size
2. Objective aperture size (smaller aperture increases contrast)
3. Spot size
4. Beam diameter on the screen
e. Example conditions: 150m condenser aperture, 43000x, spot size 4.
4. Set up Focus mode:
a. Click the Focus button.
b. Set the magnification to ~100,000x to be able to accurately focus and assess for astigmatism.
c. The diameter of the beam is not a factor here as long as the beam is not spread so far that it doses
the exposure area.
d. Using the same spot size in all three modes decreases the condenser hysteresis effect (condenser
lens remembering the previous electromagnetic strength used).
e. Example conditions: 150m condenser aperture, 98000x, spot size 4.
5. Align the Search and Exposure mode positions:
a. In Exposure mode find an object of interest that will be visible in Search mode (a piece of hexagonal
ice works well).
b. Center the object in the middle of the main viewing screen using the joystick.
c. Go to Search mode and
move the object of interest into
the center of the main
viewing screen using the
Multifunction knobs (MF). You
can also defocus the
Search mode (several
hundred microns) to
increase the contract seen with
the sample. Re-center the
object using the MF knobs.
d. Check the alignment of the
Search and Exposure
modes by finding another
object of interest (or a hole) and
adjusting the alignment of
Search mode if necessary
(using the MF knobs).
6. Set up Focus positions, 1 & 2:
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a. Center a hole from a Quantifoil grid in Exposure mode.
b. Select 1 under the Focus button on the Low Dose window.
c. Open the flapout window and click on the settings tab.
i. Look under the Focus section and make sure that the Mode only is selected.
ii. Click the reset button. This should bring the Focus 1 setting to the Exposure mode setting.
iii. Decrease the magnification to ~16,000x to see the holes in the carbon film on the Quantifoil
iv. Using the MF knobs (X moves distance out, Y moves rotation/angle) to position the Focus 1
setting on the carbon in between the holes of the Quantifoil grid. Increase the magnification
back to 98,000x and center and spread the beam so that it is on the main viewing screen.
v. Increase the magnification back to ~100,000x and center the beam.
d. Select 2 under the Focus button on the Low Dose window.
e. Repeat step c above, but set the angle of Focus 2 to be ~180 away from Focus 1 (Focus 1 will be on
one side of the exposure hole and Focus 2 will be on the opposite side of the Exposure hole).
7. Click on Search to return to low magnification.
8. Find a good area of ice and center the hole in Search mode. Go to Exposure mode and adjust the beam
centering and diameter if needed.
9. Go to Focus mode focus the image.
a. Reset the defocus value to 0.
b. Click on the other Focus mode setting (1 or 2).
c. Focus the image.
d. Look at the defocus value.
i. If the value is 0, then your grid is flat and you can defocus to the desired amount.
ii. If the value is not 0, then your grid is not perfectly flat. Take the value and divide it by 2. This
should be approximately where the exposure area is located. Turn the focus knob to go to
this value. Now reset the defocus and defocus to the desired amount.
10. Make sure that your sample is not drifting (no visible movement).
11. Click Blank to activate the beam blanker (yellow). Click Exposure mode, then lift the screen.
12. Take an exposure by clicking Blank (turns grey) and then taking an exposure.
13. Lower the screen and check/adjust the beam centering and diameter. Also, check the hole centering and
adjust with the joystick. If the hole centering is adjusted, then return to Search mode and adjust the centering
with the MF knobs.
14. Select the next good hole in Search mode and repeat steps 9-12.
15. When data collection is complete, return to Search mode. If desired, save the low-dose conditions in a file for
future use (Low dose window, flapout window, settings tab).
16. Click Low Dose button to close low dose.
17. If finished with microscope session, then do normal shut-down procedure.
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