Operating Procedure for Tecnai G2 STEM

(Li Yang, Version 1.7)

General Reminding from Li:

1. Tecnai G2 Scanning Transmission Electron Microscope (STEM) is an imaging/analysis
instrument at 4D/Nanoimaging. Being upgraded in Oct. 2013, it has been serving users
at SFU for 11 years, in both research and teaching with its multifunctional applications.
I’ve been managing the machine routine running, training users and helping
them on site since 2003. Given enough time and efforts, every user will get the most from
operating the complicated instrument by themselves. The standard operation manual
(SOP) presented here is written for 4D/NI web site, dedicated for guiding trainee in
Phase 1 and 2 to use the STEM effectively. For both users studying efficiency and the
instrument safety, the SOP is divided into three parts. Please keep me informed your
concerning before trying out anything you are not sure on the STEM, and pay special
attention to the content in bold font in the SOP.

2. For efficiency of the training, new trainees are strongly advised to:
a. Print out the SOP and take it with you during your training. Read it before Phase 1
training. Get concerning TEM knowledge in advance by internet or taking
a course. Always clarify your questions by either making an appointment meeting with
me (<0.5h in 9:00-15:00 in my work day), or discussing on site in your slots.
b. Arrange a period time with me in your Phase 1 for Phase 2 in advance for intensified
practice on the Tecnai G2. During Phase 2, clarify technical details concerning
instrumental and operations.
c. During Phase 2 and Phase 3, discuss applications to your research projects to clarify
technical concerning such as the proper setting of parameters dedicated to samples,
data acquiring and processing.
The time necessary for each phase varies depending on users situations. Look into
your STEM plan and discuss it with me as soon as possible. It takes time to put your plan
on-line since it needs work coordinately with both my availability and the tool occupancy.

3. For the effective work on the STEM, researchers are strongly recommended to
a. Take notes and revise the SOP according to your situations. Write your own
operational manual based on the SOP and the details of applications dedicated to
your projects by the end of phase 3 training.
b. Book a slot for the proof reading of users’ own operation manual with me on the Tecnai
G2 during your phase 3 training for clearing any questions on site. A User may be
granted full access to Tecnai G2 , means be able to book on-line and work in
after hours and weekend to get large amount of data after passing the qualification
test with his own SOP accepted by me in advance.
c. Book a refresh slot working with me on the STEM if users stop using it >2 months.

4. For my serving’s efficiency, I appreciate to get users’ proposals (or users own manual)
2 week in advance of their on-line booking for
a. Refresh of instrumental, operations
b. Tutorial of applications and application adaptions to their research projects.

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 Part I Conventional Transmission Electron Microscope (CTEM) Imaging
and Selected Area Electron Diffraction (SAED)

Condenser
C1 Aperture

Condenser
C2 Aperture

Objective
Aperture
Select Area
Aperture

Fig. 1 Picture of part of Tecnai G2 STEM: column and control console

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Pre-test check:

1. Clarify the functions of the lens and apertures with Li in Phase 1 training.

2. Before you operate the STEM, confirm below instrument conditions:

a. The position of the apertures (indicated by a pin on the ring):
C1 is at #4, C2 at #4 or #3, Objective Aperture is out at #7. Selected Area Aperture is off axis:
lever bar to the right at #4.
b. The Single Tilt holder is in the stage.
c. Confirm the system lights on the console are illuminated as shown in Fig. 2. If not, stop, consult Li.

Fig. 2 Normal state indication of the Lights on the console of Tecnai G2 STEM.

Starting up

1. Log into your user’s account.

2. Start the software one by one following the sequences: Filter Control, Digital Micrograph, Tecnai
Imaging & Analysis, Tecnai User Interface. Start the following one after the former finishes initializing.
3. Make sure that vacuum in Gun, Column and Camera chamber are within limits (values on a GREEN
background) in the workset (the Workset is at "Setup" tab, "Vacuum" widget, as shown in Fig. 3:
Gun =1, Column < 10, Camera < 30).

Fig. 3 Vacuum widget shows the column valve is closed

4. Fill the Liquid N2 canister and place it on the supporter under the cold finger: Hold the canister properly
while inserting the cold finger's Cu wires into Liquid N2. Too fast will make Liquid N2 to boil and spill, do
it slowly.
Trainee should get an orientation of how to do it safely with Li. However, ask for Li’s help if you
are not ready to do it by yourself.
The Liquid N2 canister should be re-filled every 3hrs to keep the column vacuum safe.
The Column Log data will increase dramatically if the canister is empty.

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 5. Bring on the Vacuum Overview window. Make sure that column valves (V4, V7) are closed. If not,
close them with the "Col. Valves Closed" button on "Setup" tab, in "Vacuum" widget.

Fig. 4 Vacuum Overview shows the column valve (V7 and V4) is closed

Note: The version of the user interface of the Tecnai G2 is 4.4.1. FEI's microscope control software tries to
emulate the behaviour of real control panel (buttons with lights), rather than using the common GUI
conventions. Sometimes, this might be misleading. Like on a real control panel, the text of a button will be
the same all the time, regardless of the current context. What changes with the context is the button's
colour. In FEI's software, when the function is active, the button is yellow (status = true/ON); when the
function is inactive the button is grey (status = false/OFF). If the button's text is greyed out, the button is
actually disabled / inactive regardless its colour, and the status of associated function can’t be changed by
the user. Example: the text on "Col.Valves Closed" button stays the same even when the valves are open
- it describes the function associated to that button, not its action. The current status of the function is color
coded: when the valves are open, the button is grey ("valves closed" = false); when the valves are closed,
the button is yellow ("valves closed" = true).
Report anything wrong observed in detail to Li Yang on time is the most effective way to
get it fixed. Operators are not eligible to do any trouble shooting.

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Sample loading / switching

1. Remove the holder only when the stage LED is not bright (A holder should be always kept on the
stage).
2. Do use two fingers to press against the purple plate while pulling the rod to keep the stage alignment
3. Pull rod out slowly and continuously until it reaches a stop. Keep it straight, don't wobble.
DON'T LET IT GO till it reaches the stop, hold it at all times. Rotate the rod clockwise a little at the stop.
4. Rotate the rod clockwise ~120 degrees until it reaches another stop. You may release the handle and place
your fingers at a better position while rotating, support the rod with another hand.
However, do NOT reverse the motion.
5. While pressing the purple plate with two fingers, do the last pull. It's a fairly strong but short pull
following by a quick release of the rod, so do it slowly, firmly but gently. Then remove the rod from the stage
horizontally and slowly.

Load sample to the holder

Note: 1. Never load magnetic sample to Single Tilt holder! Make sure during your Phase 2 training
your sample is exactly fit for the selected holder.
2. Avoid any interruption during the sample loading process.
3. Work with gloves on when you loading the sample to the holder.
3. You may ask Li to help you loading the sample till you are confident to do it by yourself.

Before the sample is loaded,

1. Use the the optical microscope to check the cleanness of the holder, paying special attention to
a. The holder end area that will be in the column.
b. The o-ring of the rod
c. The little pin on the holder that carries the code of the Compustage.
Make sure No lint or other dust on them, and they are in good state.

2. During loading the sample to the holder,

a. For Single Tilt holder: No magnetic sample to it! Make sure both the sample and press ring is in correct
position/orientation, and is secured.
b. For Double Tilt holder: Make sure the sample, washer and screw ring is in correct position/orientation, and
is secured. Below is the figure of the double tilt holder:

Fig. 5 Double Tilt holder of Tecnai G2 STEM

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3. Before moving the holder to the stage, double check the cleanness and the security of the holder.

Loading the sample holder to the compustage

1. Align the tip pin on the rod to the "Close" line on the purple plate.
For the Single Tilt holder, the arrow on the black handle should point toward the mark on front of the
Stage housing.
2. For the Double Tilt holder, insert it and push the rod until it reaches a stop and the end of the cord on the
holder keeps at a horizontal position. The red LED indicator comes on automatically (in the lower left side of
the sample holder housing), meanwhile the "Turbo on" button (in workset, "Setup" tab "Vacuum" widget)
becomes orange and pumping starts. The message "Pumping specimen airlock" and the remaining
time will be displayed in the "Vacuum overview" window.
3. Select the sample holder type in the Microscope Control software. Wait for:
a - "Turbo on" button (in workset "Setup" tab, "Vacuum" widget) to become yellow and active, and
b - the message "Pumping specimen airlock" to disappear in the "Vacuum overview" window.
c - the red LED indicator on the sample holder housing to turn off,
4. Hold the rod and rotate it counter-clockwise for ~ 120 degrees until the large flat pin on the inside of rod's
handle aligns with the hole under the rod and the arrow on the handle points vertically downward. At this
point you MUST HOLD THE ROD FIRMLY to prevent it from being pulled in brutally by the chamber's
vacuum.
5. While holding the holder rod, let it slowly slip into the sample chamber which is in the column.
6. Turn turbo pump off by pressing the "Turbo on" button to grey.
7. Put the stage cover over the sample holder head. Never extract/insert holder with beam spot visible on
screen.
8. Column pressure may increase after sample loading. Wait for it to decrease to 10 or below before opening
column valves. The first loaded sample on an outside column holder may take a little longer time.
9. While waiting, without open the col. valve, move your sample around, watching for any changes of the
col.log data. Move on to next step if no change. If it increases dramatically at some locations, stop there
and wait. If the bad situation repeats, stop your test and report the situation asap.

Fig. 6 Control panel of Left Hand (LHP) and Right Hand (RHP) of Tecnai G2

Beam initial tuning

Note: : a. This operation aims at getting a workable beam.

b. LEFT track ball moves the beam; RIGHT track ball moves the sample.

1. Clarify the position of the apertures to prevent them from blocking the beam: C1 is at #4, C2 at #4 or #3,
Objective Aperture is out at #7. Selected Area Aperture is off axis: lever bar to the right with pin at #4.
2. Check if the beam settings are exactly yours: Acceleration voltage data, on, or off. Extract voltage data,

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 FEG state, on or standby; Gun lens #, FEG emission current.
3. Confirm the col. log < 10. Open column valves by pressing the "Col. Valves Closed" button (in the
"Vacuum" widget). The button on the monitor will turn from yellow to grey with a beam spot showing up on
the phosphor view screen.
3. If you see the beam, move on to step 4. If the beam spot does not show up, you may search for it by
a. Decrease the magnification till it swings back into the screen.
b. Move gently with RIGHT track ball since the bars of the sample grid may block the beam.
c. Load the most recent alignment file of supervisor.
If the beam is still not available after all the above efforts, closed Col. Valve and report the
situation to me asap.
4. Set magnification to M4400x. Move the beam spot to the view screen center with LEFT track ball.
5. Use "Intensity" knob on the left panel to focus the beam.
6. Adjust C2 aperture centering knobs to get the beam to be concentric to the center of the view screen.
When no shifting of the beam during the intensity knob goes through a cross-over, the beam is set properly
to illuminate on the sample.

It’s good time to change magnification to low magnification (LM) range to locate your interest sample
area and save the position in the stage map.

Adjusting sample to eucentric height

1. Keep the magnification at M 4400x, find a feature near your interested area.
2. Coarse adjustment:
a. Press the ‘Eucentric focus’ button (it is on right panel) to have the objective lens to down load its
eucentric height preset.
b. Press L2 to reveal the wobble range the feature, Press L2 again to stop the wobble of the stage.
c. Type an data in the Z (it is on “set” tab of flap out of "Stage2" widget in the Workset, "Search" tab), and
click on ‘go to’ below, keep trying till getting the minimum contrast of the feature.
3. Fine adjustment:
Press the Wobbler button next to ‘Eucentric focus’ button. Minimize the distance between the two apparent
images with Z axis height control touch button gently. A minimum contrast should appear when the two
images are overlapped. Now, the feature position is adjusted to ecucentric height. Switch the wobbler off.

Note:
a. The Eucentric Focus of objective lens is set to the col. alignment with different accelerating
voltage, eg. for 200kV, Obj% is nearly 90.70%. The beam will shift a lot if it is not the case.
The bad situation will disturb the sample stability.
b. The feature of the sample will show minimum contrast and minimum shift (with stage tilt A )
when it is set to eucentric height.

Basic Beam Alignment

Operators are not necessary to do the column alignments. After get the beam properly illuminated on their
sample, and set the sample at eucentric height, they may move on to test on their samples quickly after
finishing the sample sensitive ‘direct alignment’ step by step listed below.
1. Make sure that the objective aperture is not inserted in with the pin at 7.
2. Keep magnification to M4400x.
Adjust beam intensity ("Intensity" knob on left panel) so that the beam image is visible.
3. Use "Direct Alignments" widget, which is in Workset - "Tune" tab.
4. Select "Beam tilt pp X" in "Direct Alignments" widget.
5. Use MF (Multi Function) X/Y knobs (left/right panels) to adjust the beam until the beam image stops shifting
(the beam and sample image will wobble all the time, but the beam image circle shouldn't shift.).
6. Select "Beam tilt pp Y" in "Direct Alignments" widget and repeat the procedure 5.
7. Select "Beam shift" in "Direct Alignments" widget. Centre the beam only by MF X/Y knobs.
8. Select "Rotation center" in "Direct Alignments" widget. Use MF X/Y knobs (left/right panels) to adjust the
beam until the feature image stops shifting - sample features keep changing like they're popping up/down.

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 You can use the small focusing screen and the optical microscope to view better and tune it even finer.
9. Press ‘Done’ when each tuning is finished. The new setting will be saved and notified.
Note: Use only MF (Multi Function) X/Y knobs to centre the beam while do ‘Beam shift’ in ‘Direct
Alignments’. Never use the left track ball. Wrong operation will lead to beam disappearance
especially at higher magnifications.

Basic image tuning using the phosphor view screen (for working effectively):

1. A projection of electrons through the membrane of the TEM sample will show up on the phosphor view
screen after the above operations.
2. Focus the image: You can focus visually using the phosphor screen and the “Focus” knob on the right
panel. If you want a better precision, use the small focusing screen and the optical microscope attached to
the observation window. Bring in/take out the small focusing screen by rotating it's lever located on the left
side of the viewing chamber. The focus knob has an outer ring that control the focusing sensitivity by
setting the focus step.
Note: Be careful while working at higher mag: a large focus step (>4) will produce a large shift at a
touch of the focus knob - the beam will go away! It is very easy to rotate by error the focus step
knob instead of the focus knob itself.
3. Identify the focus state of sample grains by observing the out line surrounding them:
a. White line: defocus
b. Black line: over focus
c. Focus: At this focus state, amorphous membrane will display minimum contrast; crystalline grains will
reveal fine details.
4. Diagnose the objective stigmatism on the view screen by focus knob:
Stigmatism is the distortion of lens. Objective stigmatism makes the image blur in both defocus and over
focus state, and make the out line appearing not even: one side is white while another side is black. At this
situation the minimum contrast image is unable to reveal the image details sharply. Objective stigmatism
can be corrected effectively by making using the DigitalMicrograph Software and Gatan CCD. Operations
will be described later in High Resolution Transmission Electron Microscopy.

Conventional Transmission Electron Microscope imaging

A 4kx4k Ultra-scan CCD camera is equipped on Tecnai G2 for recording imaging. It is run by DigitalMicrograph
software (version 1.85.1535). The DM is also used for image optimizing during data acquiring and processing.

A. Bright/Dark Field Transmission Electron Microscopy (BF TEM /DF TEM):

BF TEM is the TEM image when the scattered/diffracted beam is blocked by objective aperture.
DF TEM is the image with scattered beam/diffracted beam selected by a hole in the objective
aperture. Both BF TEM and DF TEM operations are often used to observe grains morphology inside
the samples.

1. Set the electronic optical axis for centering the objective aperture:
a. Press "Diffraction" button on right panel after the sample is focused. A diffraction pattern will show on the
phosphor screen with a brightest transmitted beam spot near the center area.
b. Use MF X/Y to centre transmitted beam spot. If beam spot is not round press "Diffraction" button
in "Stigmator" widget and use MF X/Y to adjust stigma. The transmitted beam spot reaches minimum size
when the stigmatism is corrected. Press "None" button in "Stigmator" widget to return to beam centring.

2. Insert the Objective Aperture to get BF TEM image:

While in diffraction mode, rotate the ring of the objective aperture to insert it with the pin point to a proper #.
Aperture #4 is the smallest aperture hole, inserting it will mostly enhances the contrast. Spread the beam
with "Intensity" knob to reveal the edge the aperture hole. Only when the aperture hole is visible, centre
the aperture with the aperture position control screws on the objective aperture.
When done, press "Diffraction" button on RHP to revert to Bright Field TEM imaging.
BF TEM are often used to observe grains morphology, which may be applied to screening the reaction
process, studying samples on the relationship between the structure and the properties, etc.

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3. Insert the Objective Aperture to get DF TEM image:
DF TEM is the image with scattered beam/diffracted beam selected by a hole in the objective
aperture. There are generally 2 types DF TEM image: on axis one and off axis one.
a. On axis DF TEM image is tilt the beam or the grain to move the interested diffraction spot to the view
screen center to let it go through the hole of the inserted objective aperture.
b. Off axis one is to select related electron diffraction spot by directly circle the interested diffraction spot with
the hole of the inserted objective aperture.
DF TEM image is used to study the defects in crystalline samples, the morphology of the
participated phase in the sample, etc. Users may chose the type of the DF TEM image regarding sample
and/or instrument conditions.
Li strongly recommended that trainee / researcher to work with her on site to figure out an operational
approaching dedicated to his project when DF TEM imaging is necessary in his Phase 2 training.
Note: a. Tecnai G2 here is equipped with a super twin objective lens for its high resolution. There are
actual 2 series of Objective Aperture holes on the OA holder.
b. Center the holes with the knobs only when the hole is visible.
c. Chose the proper hole # to your application during your Phase 2 training with Li.
d. You may do Dark Field TEM imaging to study the second phase grains or crystal defects
by selecting concerning diffracted beam go through. Consult Li on site in your slot with the
details prepared in advance.

B. Electron Diffraction

There are 2 ways on the Tecnai G2 STEM to do electron diffraction. Both are commonly used for setting up
the electronic optical axis for BF/DF TEM imaging.

1. Convergent Beam Electron Diffraction (CBED):

a. Chose interested grains use the beam.
b. Press "Diffraction" button on right panel after the grains are focused.
c. Focus the diffraction spot with ‘focus’ knob.
d. Remove the stigmatism by tuning Mfx or Mfy with ‘diffraction’ selected in ‘Stigmator’. Click ‘none’ to
finish.
This way is mostly applied to grains of tens nanometers.

2. Selected Area Electron Diffraction (SAED)

a. Focus the interested grain.
b. Adjust the ‘intensity’ knob spread beam to full phosphor screen.
c. Insert the selected area aperture by turn the lever on the SA aperture holder from right to left.
Locate and center the hole with the screws on the aperture.
d. Press "Diffraction" button on right panel.
e. Focus the diffraction spot with ‘focus’ knob.
f. Remove the stigmatism by tuning Mfx or Mfy with ‘diffraction’ selected in ‘Stigmator’. Click ‘none’ to
finish.
This way is mostly applied to study large grains regarding crystalline characterization of single crystal and
defects in it and grain boundaries.

Note: During their Phase 2 training, users need to have a specific safety orientation with Li to clarify
a. How to operate the SA apertures since a biprism has been installed on the SA aperture holder.
b. How to operate CCD camera and the beam stopper. The beam stopper must be inserted to
block the transmitted beam when the SAED pattern is imaged by CCD camera.
c. How to read the diffraction patterns, and process the data accurately.

C. High Resolution Transmission Electron Microscopy (HRTEM)

High Resolution Transmission Electron Microscopy is widely used to study the crystalline state of grains
in nanometer. It’s in fact a coherent image of the transmitted beam and diffraction beams of the interested

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 grains.

To get the sample HRTEM imaged,

1. Get the objective lens tuned well with properly focused on the sample, and stigmatism corrected:
a. Increase the magnification by rotating the Magnification knob to 250kx or even larger magnification while
keep proper intensity to view an amorphous area near the interested grains in the center of phosphor
screen. Keep it near to focus.
b. Rotate the objective aperture ring to get the aperture completely out with the pin# at 7.
c. Adjust the ‘intensity’ knob to spread the beam to the full phosphorscreen, or even more dimmer.
d. Press R1, observing the phosphor screen to lift up, and get the image using the search in DM.
e. Drag down the menu of Process in DM, select ‘Live FFT’
f. If the FFT image is not circular, select ‘objective in ‘stigamator’, using MF knobs to get it round. Click ‘none’
to finish. Close FFT, return to image. The image should be sharpen with more details revealed.

2. Revealing / optimizing HRTEM image.

a. Return the interested grains area to the center of viewing field, fine tuning focus knob with smaller # focus
step selected by rotate the ring of the focus knob.
b. Fringe or dot pattern will appear in crystalline grains if they are orientated properly.
c. Finely tuning the focus while using the CCD camera directly on computer's monitor. Select most interested
area on the grains to image.
Note: It is proved more effectively
a. Using the CCD camera for optimizing the imaging than get the initial focus.
b. Correct the stigmatism of objective lens with CCD camera / DM, then image the sample.
c. Important tips for safely using the CCD camera:
1) Always spread the beam ("intensity" knob) so that you have a dim beam to avoid saturation of the
CCD detector.
2) Always switch the camera back to the phosphor screen by press R1 before you need to focus with
large focus step, or decrease the magnification, or in both the cases.
3) Always adjust the contrast of ED pattern properly with inserting the beam stopper before
recording it with CCD. Practice it with Li to set up your operating protocol with your sample
if you are not sure.

Fig. 7 Adjust exposure time and Take a picture with camera window of Digital Micrograph

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Taking pictures, saving and transferring the data

1. Take the picture by pressing the ‘ Start Acquire" button in the Camera window of DigitalMicrograph.
2. Save the picture right away.
Default graphic format of DigitalMicrograph is dm3, a proprietary format of Gatan, Inc. Given enough time
and space, You’d better save your image as *. dm3 as the original data. The data are saved remotely on the
data computer to keep the capacity of machine control computer. It takes a few seconds to complete the
saving operation over the network, please be patient.
3. Delete the picture display on the screen asap after it is saved.
Pictures kept on the monitor may drain the capacity of the computer and make the controlling knobs
of the EM to work in an abnormal way.
4. Transferring, backup and delete your data asap is the secure way to keep your data safe.
Your data will be deleted in 2 weeks. It may be damaged by something wrong of the computer.
5. Process your data using the off-line DigitalMicrograph on the data computer in TASC2 6120.3.
Never use ‘Save as’ to transfer the *.dm3 to *.tif. Such a 36 bit *.tif cannot be opened by Photoshop
and DigitalMicrograph.
Select ‘Save Display As’ to transfer the *.dm3 to *.tif or other available image format if necessary.

Fig 8. Correct way of transferring *.dm3 to *.tif.

Finish operations

1. Quit you applications. Return the microscope state as you find it: BF TEM image mode with a beam visible in
the center of the phosphor view screen at Mag. 125X. HV at 200kV, and is on. Objective lens is activated.
2. Return the aperture hole positions to C1 at #4, C2 at #4 or #3, Objective Aperture is out at #7.
Selected Area Aperture is off axis: lever bar to the right at #4.
3. Bring out Vacumn Overview, close the column valve by pressing the "Col. Valves Closed" button (in the
"Vacuum" widget).
4. Click ‘holder’ in “Stage” widget to set sample position index (x, y, z, α,β) all to zero.
5. Confirm the red LED on the stage is off. Extract the holder, and unload your sample.
Do not extract the holder if the red light on the Stage keeps bright. Check if β=0. If not, set it to by
click on “holder” or “ AB”. If it cannot be set to zero, STOP. Report the situation to Li asap.
6. Load the empty ST holder to the stage.
7. Check the website to see if you are the last user of the day, situation may change while you are working on
the STEM. Re-fill the LN2 canister if the following user will show up in 3hrs.
8. Do ‘cryo cycle’ if you are the last user.
Take off the canister exactly after you heard that the turbo pump starts well, and put it on the ground
gently close to the wall for its safety. Forget to take it off will lose the beam when the next user

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 starts.
9. After the cryo cycle starts well, turn off the imaging software in the reverse sequences when you initialize
them:
TIA, DM. You may click ‘Yes’ to ‘Do you want to save changes?’ if you satisfy with them. Or, select ‘No’.
When you close Filter Control, always chose ‘NO’ to answer ‘Do you want to save the changes to
GIF 2000?’
The last software to close is the user interface.
10. Keep user record log to the lab log book as listed, identifying the holder used as ST, or DT
11. Transferring, backup and deleting your data asap since that is the most secure way to keep your data safe.
Your data will be deleted in 2 weeks. They may be damaged by something wrong of the computer.
12. Log off both the Tecnai computer and the data computer to keep your alignment conditions and data safe.
NEVER SHUT DOWN the Tecnai G2 computer when you finish.
a. It needs to be ‘on’ always to keep all the systems of the electron microscope working.
b. For the instrument safety, users are not eligible to not turn off / on the computer and the electron
microscope. Shut it down accidentally will damage the STEM, costing money and taking time to
bring the instrument back to spec.
13. Clearing all your belongings, taking them away with you. Turn off the monitors and illuminating lights in the
slab. Make sure the room door of TASC2 6120 is locked behind you when you leave.
14. Do the ticket to 4D/NI each time a user accesses the STEM following instructions from me. Clear
questions with me concerning the ticketing no later than Phase 1 training.

Thank you for your supports to my work and the facility running!

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 Part II Analytical Electron Microscopy (AEM) I
Scanning Transmission Electron Microscopy (STEM)
And Energy Dispersive Spectrum (EDS)

Reminding from Li:

1. The operations of STEM and EDS is more demanding regarding the parameters
controlling over the specimens. For their work effectively, trainees need to satisfy both
conditions below:
a. Qualified users who finished Phase 3 of Part I.
b. Had at least 20 hrs independent TEM working experience on Tecnai G2 after
finishing Phase 3 of Part I

Introduction:

When electrons in STEM go through the specimen, their interaction with the membrane will generate signals
which contain information about the actually illuminated part of the specimen. By scanning the specimen
surface with the focused beam and collecting those signals, the Tecnai G2 STEM becomes a multi-functional
analytical instrument:
a. A Scanning Transmission Electron Microscope (STEM); It collects the transmitted beam pixels by pixels.
Depending on the detector’s collecting angle, the STEM can do the BF, DF and High Angle Annular
Dark Field (HAADF) imaging. The final one is good at telling the difference of the atomic number
(Z- contrast) if the sample thickness is suitable. The software on the Tecnai G2 to get the functions operated
is Tecnai Imaging and Analysis (TIA, version 4.5).
b. An Energy Dispersive X-ray Spectroscope; It collects the X-ray emitted from the scanning spot pixels
by pixels. It is widely used to detect the elements in interested area of the specimen by spot detecting, line
scanning and element mapping. Tecnai Imaging and Analysis and EDAX are the 2 software on the Tecnai
G2 to get function operated.
c. An Electron Energy Loss Spectroscope; By studying the energy loss of the electrons in the transmitted
electron beam using an energy prism and filter, the EELS can reveal both the information of elements and
chemical bounding inside. The technology of imaging by selecting of different energy range is EFTEM since
it is done by choosing proper slots with different width in an energy filter.
This part of operation will be introduced separately in Part III due to its complexity in both data collection and
data procession. The software on the Tecnai G2 to get the function routine operated are Filter Control and
DigitalMicrograph.

Requests of STEM and EDS test:

1. Specimen must be clean, dry, thin enough, preferably be chosen from those had been studied by TEM.
2. Specimen must be loaded in the suitable holder, preferably the DT, with specimen side facing the electron
beam.
3. Clarify gun and column alignment condition during Phase 2 training, and keep it updated with me.
4. Clarify proper setting of test parameters over your specimen during Phase 3 training of Part I, or refreshing.
.
Setting up:

1. Find an amorphous area near your interest area (better with recognizable features), and tune the TEM well.
a. Set specimen at eucentric height
b. Correct stigmatism of both Condenser lens and Objective lens.
2. Got a BF TEM image of your interest area.
3. Remove the objective aperture by rotating the ring, confirming it is out with the pin at #7.

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STEM imaging and optimizing:

Fig. 9 STEM controlling in Tecnai User Interface

1. Activate STEM, as shown in Fig. 9

2. Load proper *.FEG as instructed on site if necessary.
3. Insert HAADF detector.
4. Click ‘search’ to start the probe scanning.
5. Click Focus on Monitor and select proper contrast area. Focus specimen reasonably well with Focus knob.
6. Optimizing Focus and Stigmatism with observing Ronchigram on the phosphor screen:
a. Move the amorphous area into the field of view.
b. Drag and place the Beam Position Marker Tool in TIA on to the amorphous area.
c. Stop ‘search’ by clicking on it again.
d. Focus, stigmator (C2) and precisely center the condenser aperture based on perfect the Ronchigram
appearance.
7. Acquire an image by pressing ‘Acquire’ on the monitor.
8. Switch to TIA data processing window, save the image as *.emi. The data can be exported as *.tiff when
necessary.

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EDS analysis with STEM:
2
1. In Stage , type 15 into α tilt to set specimen +15° to make the X-ray collected more effectively.

Fig. 10. Comustage controlling page in Tecnai User Interface

2. Get a sharp STEM image.

3. Switch to TIA Analysis window

4. Point analysis:
a. Insert EDX detector by click ‘in ‘ in RTEM Control, the ‘in’ button will turn to red color.

Fig. 11 EDAX control interface, as shown is the out state of detector

b. Acquire a STEM image.

c. Drag and place the Beam Position Marker Tool in TIA on to the spot to be analyzed
d. Click “Acquire” in the EDX tag to get the EDS spectrum.
e. Save the spectrum data in TIA data processing window as *.emi file. The data can be exported as *.txt
when necessary.

5. Elements line scanning

a. Acquire a STEM image.,
b. From the STEM image frame, select an area for drifting control by click on the square box icon
in TIA.

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 c. From the STEM image frame, drew a line on the interested feature with the line icon in TIA.
d. Under "Experiments", as shown in Fig. 12, and "Select Component", choose "Spectrum Collection"

Fig. 12 Experiments for set up EDX line scanning and mapping

e. Under "Select Experiment", choose "Drift Corrected Spectrum Profile".

f. Set up the scanning parameters on the expanded flap:
Profile size 10-100
Dwelling time 2000-10,000
In Collection setting:
choose "yes" for acquire EDX spectra
choose "yes" for acquire STEM images.
Elemental Process, choose “No”.
In Correction Setting: Number of acquisition in slice 2, reference image size (X&Y) 64,
Reference Image dwell time (us) 10. Number of acquisition in slice 2, Predict draft rate choose
“ Yes”. Set the correlated filter start as 0.05 and end as 0.80. Then highlight the drawn line on the
interested feature.
g. Click "acquire" in "Experiments" to begin collect the X-ray signal.

6. Element mapping:
a. Under "Experiments", and "Select Component", choose "Spectrum Collection"
b. Under "Select Experiment", choose "Drift Corrected Spectrum Image".
c. On the expanded flap:
set image size (X) as needed. This is the number of slices along the X-direction
choose "yes" for acquire EDX spectra
choose "yes" for acquire STEM images.
d. Draw a box on an area away from the analysis that contains a feature edge. This will be used
for drift correction and should not be to close to the edge of the frame.
e. Draw another box on the area to be analyzed by EDX mapping.
f. Click "acquire" in "Experiments" to begin collecting the X-ray signal from the interested area.
Note: All the parameters settings above need to be modified when applied to different specimen.

Image processing:

1. Save the data in TIA as a *.emi file.

Note: it is very important to practice with TIA tutorials to master the skill in addition to following the
brief guideline.
2. Click "Setup" under Automap and choose "Integrated Intensities"
3. Click "Generate Output" under Automap to produce individual element maps.
4. Save each window by highlighting the image, and choosing File -> Export Data -> Export as .tiff
with scale marker.

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Spectra processing:

1. Open any element map.

2. Place a marker at a point for analysis.
3. Hold the 'alt' key and drag the marker to the EDX spectrum, and drop it on top of the blue histogram. The
EDX spectrum associated with the pixel at the marker position will be displayed.

Finish operations:

1. Extracted EDAX detector by click out in RETEM.

2. Click on “STEM” to turn it gray.
3. Get a beam and center it on phosphor view screen.
4. Close the column valve.
5. Continue to carry out other finishing operations listed in Part I.

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 Part III Analytical Electron Microscopy (AEM) 2
Energy Filter Transmission Electron Microscope (EFTEM)
Electron Energy Loss Spectrometry (EELS)

Introduction

The Tecnai G2 STEM is equipped with Gatan Imaging Filter 2000(GIF). Its main components and functions
are described in Fig. 13. The GIF is a post column type, installed at the bottom of 4kx4k Ultra Scan Camera
2
(This is the routine camera for TEM imaging). GIF is used on Tecnai G for doing Electron Energy Loss
Spectrometry (EELS) and Energy Filter Transmission Electron Microscopy (EFTEM). The software work
for those applications is Filter Control (version 1.85.1535) and DigitalMicrograph (version 1.85.1535).

Fig. 13 Diagraph description of Gatan Imaging Filter 2001

Fig. 14 Common parts of Electron Energy Loss Spectrum

Fig. 14 describes basic parts of Electron Energy Loss Spectrum. By studying the EELS in the transmitted
electron beam from the interested area of the specimen, both chemical elements and chemical bounding
among the elements can be revealed, sample’s thickness can be measured as well.

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The image technology which chooses beam based on its energy and energy range is called EFTEM.
EFTEM has some unique advantages. For example.
a. Choosing only the Zero Loss beam to image is able to sharpen your image from the thicker area of
sample.
b. Choosing elements by choosing energy range to image is able to figure out the features challenged
to reveal in routine TEM imaging.

The EELS mapping and EFTEM is an even demanding technology since it is good at featuring from
a. Samples which are composed mainly of the lighter elements, such as biology sample, soft material
sample observed in TEM under cryo condition, etc.
b. Samples composed of elements of different oxidation state, etc.

Pre-requests of EFTEM and EELS training /test:

Due to its poor peak to background ratio of EELS, the application of the technology challenge researchers in
both instrumental and sample conditions. Please keep checking the efficiency of doing the research and plan it
in advance. Trainees need to be qualified for all conditions below:
1. They must be qualified users who finished Phase 3 of Part I and II, which enables them to adapt the
instrument conditions to investigate their specimens safely and effectively.
2. Choose only the proper sample to investigate; specimens with large flat and very thin area are preferable.
3. They need to collaborated work with me during their phase 1 and phase 2 of the Part III training to master the
co-ordinating operations of the hardware of the Tecnai G2 and Gatan Imaging Filter (GIF), as well
the software. Once fully understood how to operate the Tecnai G2 STEM with the GIF to get proper results
from the training sample, they should be very effectively to switch the approaching to their project.
4. Researchers need to keep improving their operation skill during the phase 3 training and collaborating work
with me to build up necessary database for effectively setting up EELS /EFTEM research protocol.

Preparation operations on Tecnai G2

1. Load the specimen into the Double Tilt holder properly, with specimen side facing the electron beam.
2. Load the holder correctly on to the stage.
2. With ‘Col. valve closed’, exercise the stage / holder while waiting for the col. log reaching 6.
3. Clarify gun and column alignment condition during the Phase 2 training of the Part III, and always keep it
updated when you do EELS and EFTEM tests.
Note: The real alignments may be different even you load your saved good one. Contact me for help
if it bothers you too much.
4. Clarify proper setting of test parameters over your specimen during Phase 3 training of Part I and Part II,
and during refreshing.

Tune the gun, column alignment, and locate sample positions with Tecnai G2 user interface:

1. Using double tilt holder and holey Carbon Cu grid for tuning the Gun and Column alignment conditions:
a. Download related application alignment file from FEG Register, or Alignment files to get a proper beam.
b. Confirm C2 aperture is centered well with minimum stigmatism.
2. Set specimen to eucentric height. Finish direct alignment at M4400x
3. Locate and save the positions of your interested areas, as well vacant areas closing to them with Search,
2
stage .
4. Correct stigmatism of both objective lens and diffraction lens at SA, M and LM range.
5. Get BF TEM images of interested areas. Switch the closing vacant area into the big circle of the view
screen.
6. Take out the objective aperture with the pin at #7.

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Preparations for switching from TEM to EELS and EFTEM:

Set the beam spot with diameter~5mm with intensity knob, centered it at 3 o’clock position of the small
circle on the main view screen with beam shift trackball on LHP. The proper situation is shown in Fig. 15.
Log down the C2 activating % data, keep it unchanged. Change C1 activating % by press L3 for increasing
brightness, or R3 decreasing if adjustment is requested.
Note:
a. Using R1, always keep the view screen at down positon whenever the spot illumination
is adjusted.
b. Re-locate the beam spot to the GIF aperture with beam shift trackball on LHP whenever the
beam shift occurs.

Fig. 15 The main view screen of Tecnai G2

EELS and EFTEM with DigitalMicrograph

Fig.16 AutoFilter for tuning EELS / EFTEM

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Set GIF alignments by Tune GIF

1. The AutoFilter window will be displayed in DigitalMicrograph when the software finished loading, as shown in
Fig 16. The content and description vary depending on the choice in ‘Technique’.
2. Check EF-CCD in Camera menu of the DigitalMicrograph.
The camera is a 1kx1k CCD camera located at the end of the GIF 2000.
3. In Tecnai user interface, set the mag. of TEM BF image to M2250x using the Magnification knob in RHP.
4. In AutoFilter, click ‘TEM’ in ‘Technique’, ‘Search’ in ‘View’ will be highlighted.
5. Set proper beam conditions for Use ‘Intensity’ knob on the LHP to set the beam’s dimension as ~5mm
size with enough brightness. Change spot size to smaller number by press L3 to get the beam brighter.
E.g. Spot Size 3, this spot size may also be used in EFTEM later), and set its center at 3 o’clock position
on the ~5mm circle.
Note: For the CCD work efficiently, always changing brightness by L3 or R3 with the view screen
at down position.
6. Lift the screen with R1.
7. If the image on the GIF CCD camera is evenly illuminated as on the main screen, Skip step 8 goes to
step 9 to do ‘Tune GIF’.
8. If the image projected to GIF CCD camera is not evenly illuminated, the Gain Reference of the GIF CCD
must be prepared before using the camera. Prepare Gain Reference of the EF-CCD camera following
instructions below:
a. Confirm the sample area is aside to let the beam go through a vacuum area.
b. Click on Prepare Gain Reference in the Camera menu, the procedure will be carried out in a
Programmed way with asking some questions. Here are the answers to get it through:
1) Click ‘Switch’ to allocate the camera from ‘Search view’ to ‘prepare gain reference’.
2) Click ‘OK’ to close the Prepare Gain Reference window as shown in Fig.17

Fig. 17 Parameters in the Prepare Gain Reference

3) Select ‘Yes’ if you are sure the temperature with the CCD is stable.
4) Click ‘OK’ to close the window with information as ‘ Remove specimen from field of view. Spread and
center the illumination so that it is large enough to evenly illuminate the CCD. Lift up the screen to
exposure the camera if necessary’
5) Click ‘Yes’ to agree to overwrite any existing gain image.
6) The detail action preparation will be reported lively in ‘Progress’.
7) The process ends with ‘The gain reference image has been successfully acquired and saved’.
8) Click the red idle button in ‘View’, if it changes to green ‘action’ with an image frame with evenly
refreshing grey, move on to step 10 to do Tune GIF.
9) You may need to repeat the procedure of ‘Prepare Gain Reference’ whenever uneven illumination
occurs.

9. Preparation for ‘Tune GIF’: Select ‘Show Results Window’ from Window menu to get it displayed in
DigitalMicrograph . The progress and results of Tune GIF will be reported there.
Note: the Results Window may hide due to too small a displaying window of DigitalMicrograph.
10. Click ‘Tune GIF’ in ‘Commands’ in AutoFilter. The auto-controlled process will last 5min., the operator
needs to be on site monitoring the progress, and correcting deviations if requested. For example, the
message may appear as ‘The beam’s intensity is too high, would you like to adjust or abort? ‘,
If you chose ‘Adjust’, you need to
a. Push R1 to get the view screen down, deduce the brightness by pressing R3, which will make beam
dimmer with increasing spot size number, using the ‘Beam Shift ‘track ball to keep the spot align to the
prism receive aperture.

21
 b. Push R1 again to lift the view screen, check if the beam intensity falls in the green range. If yes, click
on the message window, then press the ‘enter ‘ on the key board, the process will move on.
11. If Tune GIF finishes with report as ‘unnecessary to do further tuning’, it means Gatan Image Filter is
aligned well for doing EELS / EFTEM.
Note: a. The GIF may need to be tuned twice to optimize its function.
b. The GIF conditions may change without notice. Tune GIF when necessary.
12. Put down the view screen by press R1 when ‘Tune GIF’ completes.

Set and correct Zero Loss Peak (ZLP):

The importance of setting and correcting the ZLP position in GIF is similar to the good alignments of column to
Tecnai G2. However, ZLP may drift from time to time since the GIFis very sensitive to local changes of
electro-magnetic field.
Note: To keep your GIF conditions identical, during your EELS and EFTEM session after set the ZLP,
in TASC2 6120.7
a. Absolutely no access to i-phone or other which contain metal cell phone.
b. No change of the position of the chairs.
1. Reduce the brightness of the spot by press the R3 till microprobe spot size 10 while keeping its center
position at 3 o’clock position on 5mm circle with Beam Shift track ball on the LHP. The spot visual size
should be controlled as diameter ~5cm. Change the beam brightness by pressing R3 till its light grey.
2. Press R1 to lift View screen.
3. Get ZLP: Click ‘EELS’ in ‘Technique’, ‘turbo’ in ‘View’ and ‘Zero Loss’ in ‘Energy” will be both highlighted.
Click ‘idle’ in ‘View’, it will change to ‘active ‘.ZLP will appear, drifting back and forth, not stable as a symmetric
peak at 0kV. (If it appears as a red or yellow color peak with a flat top and a wider unsymmetrical shape, it is
saturated and useless).
Note: Below are the workable operations to fix the above troubles.
a. Always choose a vacant area to get the ZLP.
b. Set proper exposure time to get a ZLP counting (above 3000) with sharpen shape,
Note: There are 2 ways to get the proper exposure time:
a. Press ‘Alt’ in the key board together with ‘Idle’ in ‘View’ in AutoFilter, set the exposure time in
Spectroscopy Setup window. Fig. 18 shows a good case.
b. Or, closed the setup window, press ↑ or↓ in the keyboard to increase or decrease the exposure
Time.

Fig. 19 Zero Loss peak with setting proper exposure time

22
 Fig. 20 Set Zero Loss Peak exactly at E=0kV with Filter Control interface

4. Set ZLP exactly at 0kV: Go to Filter Control software

a. Type +15eV or –15eV in “Adjust’ using software Filter Control, as shown in Fig. 20, then press ‘Enter’ in
the key board. Change the data in ‘Adjust’ till the ZLP is exactly at 0kV.
b. Acquire the ZLP and log down the acquire conditions.

Get the EELS from the sample

1. From ‘Energy’, select either ‘Zero Loss’, or ‘Plasmon’, or ‘Pre-C’ for lower energy range applications.
Such as: measure the thickness of carbon film, chose ‘Pre-C’. Choose ‘Custom’ for studying elements
in higher energy range, such as different oxidation state of metals.
2. Press R1 to get the view screen down.
3. Press L3 to get spot size 5, observing spot brighter. Dim brightness by spray the beam by change
the ‘intensity’. EELS signal from sample usually much weaker compared with the ZLP.

Fig. 21 EELS from carbon film after select ‘Assign ROI as’ and ‘Zoom in the ROI vertically’

23
 4. Go to Tecnai G2 User Interface (UI) Stage2 . Select saved location of interested area, double click,
Confirming it enter into the spot. For example, carbon film. Make sure the spot aligned to the GIF
acceptance aperture.
5. Press R1 to lift the view screen.
6. Try different exposure time in “Spectroscopy Set window’ in DigitalMicrograph, get the EELS, as shown
Fig. 21
7. Optimize the EELS
With rich information preparation of sample and experimental conditions, the data processing of the EELS may
be carried out effectively following the instructions in the EELS menu. Such as assign ROI properly to remove
the background, as shown in Fig. 22

Fig. 22 Remove background from EELS of carbon based on Power Law

The EELS Atlas is a very helpful tool for data processing since it collects all the available EELS.
Users may need to build their own database by reading papers if related information is excluded in the Atlas.

Fig. 23. EELS menu

Applications of EELS

1. Measure the thickness

a. Choose proper and typical area in the sample to do the measurement. On the Tecnai G2, thickness less
than 200nm flat area is workable.
b. Get EELS of the low energy range EELS include both ZLP and plasma peaks, and optimize the EELS.
c. Measure the relative or absolute thickness following the instructions in DigitalMicrograph.

24
2. Identify Chemical Elements and Chemical bounding between the elements by EELS Altas.
a. Area selected for this application must be much thinner than the one for measure the thickness.
b. Set up proper instrument conditions to get proper EELS.
c. Process EELS following related instructions in EELS menu.
d. Identify chemical elements, or chemical bounding between the elements by EELS Atlas, or user’s own
database.

EFTEM and EELS mapping

1. EFTEM of Zero Loss Peak

a. Press R1 to put down the view screen.
b. Select ‘EFTEM’ in ‘Technique’. The slit will insert with width stated in ‘AutoFilter’.
b. Press L3 to increase the brightness until getting enough gains on the EF-CCD (refer to the one when
Tune GIF at the beginning).
c. Work with DigitalMicrograph AutoFilter, ‘Search’ in ‘View’ to image the interested area.
d. Re-align the ZLP and Tune GIF if necessary by selecting the icon in the ‘Commands’
e. Try different aperture / slit of the GIF to optimize the picture.
The EFTEM image the Zero Loss Peak from proper area of the sample would be much sharper than normal
TEM imaging since the noise form inelastic scattering is filtered by choosing the width of the slit. This
application is even more powerful for imaging low contrast samples which is composed of elements with
smaller atomic number Z, especially when it is impossible to reduce the thickness.

Fig. 24 EFTEM imaging of ZLP

25
2. EFTEM of different elements
a. Press R1 to put down the view screen.
b. Select ‘EFTEM’ in ‘Technique’. The slit will insert with width stated in ‘AutoFilter’.
c. Press L3 to increase the brightness until getting enough gains on the EF-CCD (refer to the data when
Tune GIF at the beginning).
d. Make sure the EELS of the area is tuned well. The area includes particles of interested element.
e. Work with ‘Search’ in ‘View’ to image the interested area.
f. Try different aperture / slit of the GIF to optimize the image. Get EFTEM photo with proper selection of
slit width.

EELS mapping

Fig. 25. The EELS elements mapping of carbon, interface and related settings

The EELS elements mapping function is in EFTEM interface, as shown in Fig. 25.
a. By click Map C (or Ratio @ S, C or S can be replaced by other interested elements), follow the software
instructions to set or try related parameters, the process will run in a programmed way with several
images showing up eventually finishes with the element map.

Note: To apply the EELS and EFTEM effectively to their projects,

a. Trainees / researchers are strongly encouraged to work with Li’s help on site.
b. Trainees / researchers need plan a period of time to learn and master how to work with Filter
control and DigitalMicrograph software.
c. Always select ‘No’ to close the Filter Control software.

26
Important Safety Note:
Always leave some time for your closing operations:
a. Always save your data with *. dm3 to data computer whenever necessary. And set up a backup folder
there. Transfer the data to 2 US which have been tested workable, and only use them for
the purpose.
b. Do the finish operations as instructed in detail in Part I and II.
c. Log off your account, and keep the using log of the lab.
d. Ticket to 4D/NI correctly.
e. Take your personal belongs with you, if you are the last user of the Tecnai G2 of
the day, turn the lights off and lock the door of TASC2 6120.

27