FEI Tecnai G2 F20 Manual

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FEI Tecnai G2 F20 Operating Procedures
(6/01/2017)
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1. Startup
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1. Sign-up in the microscope log-book
2. Startup the software if not already running: Start the software in this
sequence:
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1. Tecnai User Interface
2. Digital Micrograph
3. TIA (TEM Imaging and Analysis)
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3. Check the vacuum: Look at the Vacuum panel under the Setup menu. The
GUN, COLUMN and CAMERA values should look like the ones below and
highlighted in green. Gun will always be 1 Log.
4. Fill the cold-trap dewar with LN2: Cover the screen window first. Fill the dewar
until it is full. If you are the first user of the day, refill it again after 30 minutes.
Refill the LN2 at least every 3 hrs to keep it full
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5. Inserting the specimen holder
Room temperature sample
There are two things to check prior to inserting the specimen holder:
(a) Under the Search tab, in the Stage2menu, click on the flap-out menu and click
Control then Holder. This will reset X, Y, Z, and αon the stage and avoid damaging the

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holder. Wait until the red light on the Compustage is off before proceeding.
(b) Remove the objective aperture by sliding the lever to the right.
Align the specimen holder with the small pin at approximately 3 o’clock. Carefully insert
the end of the specimen holder into the stage until you reach the end. Then rotate
clockwise to about 5 o’clock whilst very gently pushing inwards. This will allow the
holder to slide in to the final pumping position. The red light on the Compustage will
turn on and the turbo pump will begin speeding up (orange button color) and in ~3
minutes reach its operating speed (yellow button color). A countdown will begin in the
Vacuum Overview window. Under the Messages/Log menu select the type of specimen
holder inserted.
When the countdown has finished and the red Compustage light has switched off, rotate
the specimen about 120 degrees anti-clockwise (as far as it will go), then slowly slide it
into the microscope. Do not let go of the holder as the vacuum will pull it in and this may
damage the stage. Once the holder is inserted click on Turbo to switch off the turbo.
Cryo sample
Under the Search tab, in the Stage2menu, click on the flap-out menu and click Control
then Holder. This will reset X, Y, Z, and αon the stage and avoid damaging the holder.
Wait until the red light on the Compustage is off before proceeding.
Remove the objective aperture by sliding the lever to the right.
Under Setup tab click on Turbo to switch on the turbo pump. The turbo pump will start
speeding up (orange button color) and in ~3 minutes reach its operating speed (yellow
button color). Under Search>Stage2>alpha toggle. Enter “-55” then click Set Alpha to
tilt the stage. Under the Setup>Vacuum>Cryo click on Prepump Airlock. Insert the
cryo-holder as above (adjusting the insertion position for the tilted stage). The turbo will
start pumping on the airlock within ~4 seconds of the Compustage red light turning on.
The countdown will begin in the Vacuum Overview window. Under the Messages/Log
menu select “ST Cryo Holder”.
When the countdown has finished and the red Compustage light has switched off, rotate
the specimen about 120 degrees anti-clockwise as far as it will go, and slowly slide it into
the microscope. Click on Set Alpha to reset the stage. Click on Turbo to switch off the
turbo and top-up the liquid nitrogen in the holder.
6. Removing the specimen holder
Reset the Stage (Search>Stage2>Reset Holder).Close the column
valves(Setup>Vacuum>Column Valves). Stand up and position yourself opposite the
compustage. Use your left-hand fingers to push gently against the purple plate of the stage
and use your right hand to slowly retract the holder outwards. When you reach a stop,
rotate the holder clockwise ~120° until it stops. Then pull out gently and parallel to the
stage axis. You will have to overcome the vacuum pull but do not pull too strongly as you
may damage the stage.

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2. Microscope Alignments
1. Eucentric height adjustment
(a) Open the column valves under the Setup menu. At a magnification of about 4000-
5000 x, find a noticeable feature on your specimen.
(b) Activate the alpha-wobbler.
Change the Z height on the right-hand control panel to minimize the image
displacement.
(c) Stop the wobbler. Press on “Eucentric Focus” on the right-hand control panel.
2. Centering the condenser aperture. Condenser:
(a) Select a C2 aperture (100um for TEM/Single particle)
(b) At a magnification of ~10,000, go to crossover and center the beam using
the left trackball.
(c) Expand the beam and make sure its expands concentrically. If not center it using the
condenser aperture position knobs.
3. Condenser lens stigmation: Go to a magnification of 135,000X and go to crossover.
Under the Tune menu click go to Stigmator and click on Condenser. Use
multifunction knobs X and Y to obtain a triangular crossover beam. Once done click
on None to exit.
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4. Direct Alignments: Under the Tune menu go to Direct Alignments
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Perform direct alignments at 50,000X and close to focus on the specimen.
(a) Beam Shift alignment: Click on Beam Shift and go to crossover. Center the beam in
the middle of the phosphor screen using Multifunction knobs X and Y.
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(b) Beam-Tilt Pivot Points (X and Y): Click on Beam-Tilt Pivot Points X and go to
crossover. Try to minimize the movement of the beam using Multifunction knobs X
and Y. Ideally the beam should look like the picture below with a single bright point
and minimum movement. If beam moves away from center redo Beam-shift alignment.
Repeat the same procedure for Beam-Tilt Pivot Points Y.
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Bad
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Good

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(c) Rotation Center alignment: Find an identifiable object in your specimen.
Click on Rotation Center and minimize its displacement using MF X and Y.
(d) Comma free alignment: Use the Orius CCD to go slightly underfocus (-500
nm). Using the live FFT correct Direct Alignments: Comma free alignment X
and Y. Make sure the alternating power spectra show the zeros at the same
frequencies.
Once you have performed the Direct Alignments click on Done to exit.
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5. Centering the objective aperture.
At a magnification of 50000x press the diffraction button on the right control panel. Insert the
objective aperture in (slide left) and using the aperture position knobs on the aperture
center it around the center of the un-diffracted beam.
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6. Objective lens stigmation.
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To stigmate the objective lens go to 50,000X, start the Orius CCD and set the focus to
slightly underfocus on the carbon support and bring up a live FFT so as you can see 2-3
Thon rings. If astigmatism is present these rings will be oval. To make the rings circular go
to the Tune tab in the Stigmator panel, click on Objective to activate the objective
stigmators. Use MF X and Y knobs, one at a time, to make the rings circular. If the rings
are not visible due to severe astigmatism either reduce the magnification or defocus further
and then stigmate as before. Once finished click on None

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3. STEM mode
(a) Find an area of interest at a low magnification (~10 k);
(b) In the STEM menu click STEM, Diffraction mode will be activated; press
the Diffraction button to deactivate the Diffraction mode;
(c) Under the Direct Alignments, perform Beam Shift, Beam-Tilt Pivot Points X/Y, and
Rotation Center (turn the outer ring of the objective focus know counter-clock wise to
stop the wobbling), correct the Condenser astigmatism (triangular shape);
(e) Press the Diffraction button to return back to the Diffraction mode;
(f) Insert the appropriate STEM detector.
(g) Choose a proper camera length (~ 100 – 150 mm for LM STEM) and spot size;
(h) Click Search or Preview to view the image, and click Focus and move the red box to
a contrasty area to adjust focus using the objective focus knob;
(i) Click Acquire to obtain an image. In the TIA analysis mode, select the image or panel
and save them by right-clicking and choosing Export Data.
4. Finishing
(a) Close the column valves (under the Setup menu, Fig. 1);
(b) Reset the specimen holder position, take it out from the microscope, remove your
specimen from the holder, and re-insert the holder back;
(c) Retract the objective and diffraction apertures.
(d) Check the web scheduler. If you are the last user for the day, remove the LN2 dewar,
place a paper towel on the dewar stand and immediately go to the Vacuum panel
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under the Setup menu (Fig. 1), click flap-out menu , in the Cryo tab click Cryo
Cycle (it takes 4 hrs to complete the process);

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Using&the&Falcon&II&on&the&Tecnai&F20&
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1. Perform!all!microscope!alignments!out!of!Low!Dose!
2. Make!sure!TIA!is!open!
3. Activate!Low!Dose!and!load!the!50K_recommended.lds!file!or!set!up!
your!low!dose!as!below:!
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At!50,000x!the!pixel!size!is!2.08Å
Search
Focus
Exposure (2 sec
integration time)
Magnification
(Detector)
1,700x (Orius)
150,000x (Orius)
50,000x (Falcon)
C2 aperture
100
100
100
Spot size
9
6
6
Defocus
-200 µm
0-4 µm
Dose Rate
(Measured exposure)
on small screen)
<0.02 e-/Å2/sec
10 e-/Å2/sec (2.7 sec)

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Appendix&
Microscope&Information&
Line!resolution!(nm)!
!0.144!
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Cs!objective!(mm)!
2.0!
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Cc!objective!(mm)!
2.0!
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Focal!length!(mm)!
2.7!
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Detector!
FEI!Falcon!II!
Physical!!pixel!size!(μm)!
14!
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