FEI TECNAI G2 F30 TWIN TEM

Training Manual

Electron Microscopy Core Facility

University of Missouri

June 2013

In Emergency, Simply Close the Col Valve and Leave

Snapshot – Microscope User Interface (MUI)

Tabs Microscopy User Interface (MUI)

Vacuum Flap out

FEG Control Menu

TIA Menu

Message Box

Status window

Snapshot - Left Hand Panel (LPH) Right Hand panel (RHP)

t 5

Pre-start: Instrument

1. Fill liquid nitrogen in the Dewar. Refill every four hours.

2. Check the following scope condition

• Left picture - One “Red” and three

“Yellow” on the Setup tab.

• Vacuum state – green, gun is 1, column is 6

• Holder position should be close to zero

for X/Y/Z/A/B.

• Apertures - Aperture” is out (lever to

the right). “C2 Aperture” is 2.

3. Fill in the log book (time, vacuum values and emission current).

Choose “FEG Register” and Pull Out Alignment File

Step 1 Step 2

Select a suitable FEG register. Click “Set” Go to flap-out of “Alignment” menu. Select a matching alignment. Select all alignment items move all items to the left column. Click “Apply”.

Do NOT rearrange the alignment

files or FEG Register files

Move all items to the left

Bring Beam To Sample

Wait until column vacuum drops below 10.

Click “Column Valve Closed” to open the column valve. The “Status”

shall change to green color with “READY”.

Click “Eucentric Focus”. The “Defocus” becomes zero nm.

Confirm “Objective Aperture” and “SAD Aperture” both are out (i.e.

obj aper. and SAD aper. lever to right direction)

The electron beam should be seen on the big phosphor screen. Turn

“Intensity” knob to spread beam to about the phosphorus screen

size. Center beam using “Beam Shift” track ball.

If no beam, lower magnification and move sample around to find the

beam.

Defocus value should be maintained not far away from zero during operation (< 2-3 µm).

TEM Alignment: 1 – Eucentric Height

There are two ways to set up Eucentric Height.

1. Preferred way –

Condense beam on the area of interest. If the

area of interest is not at the eucentric height,

there will be a halo around the bright spot on

phosphorus screen. Adjust “Z” to minimize the

halo. Set magnification to 125k and repeat.

2. Standard way –

• Start Wobbler (Go to “Search”, click the “flap out” arrow and click “Wobbler”) or personalized button.

• Adjust “Z” on the right hand panel to minimize

the sample movement.

• Set the magnification to 125k to do the fine

adjustment.

• Turn the wobbler off.

Click “Eucentric Focus” to start.

For magnetic specimen, only use the preferred

way.

TEM Alignment: 2 – Center C2 Aperture

Set an appropriate C2 aperture by turning the big knob to a numbered position. For TEM, number 2 or 3 is good.

Set magnification to 125k.

Turn “Intensity” knob on the left hand panel to condense the beam to a spot.

Center the beam using “Beam Shift” (the trackball) on the left hand panel.

Spread the beam by turning the “Intensity” knob clockwise.

Adjust the two mechanical control at the C2 aperture to move the beam back to the screen center.

Repeat the above steps until the beam remains centered.

Mechanical Control

Phosphorus screen

TEM Alignment : 3 – Condenser Stigmation

Adjust the condenser stigmation if the beam is not circular.

Go to “Tune”, click on “Condenser” from the Stigmator menu.

Use the “Multifunction knobs (MF X/Y)” to adjust the condenser stigmation in both x and y directions to ensure the beam is round and expands concentrically.

Click “None” after finishing to exit stigmator.

TEM Alignment : 4 – Direct Alignment (Gun Tilt)

Caution – if you are not familiar with gun alignments, don’t do it.

Purpose - The gun tilt makes sure that the electron beam from the gun comes down parallel to the optical axis, so that no electrons from the beam are lost before they can be used for imaging, etc.

Procedure - select a hole area without specimen. Set magnification at around 10k. Click “Gun Tilt” to activate. Adjust “Multifunction knobs” in both x and y directions to maximize the screen current (nA). Click “Done” after finishing. Correct condenser stigmation again.

Status window

TEM Alignment : 5 – Direct Alignment (Gun Shift)

Purpose - Shift the electron beam sideways so that it comes down along the optical axis.

Procedure -Condense beam to a spot and center using track ball. Select “Gun shift” to activate. Change spot size from 3 to 9, center beam using Beam Shift (track ball in LHP); change spot size back to 3, center beam using MF X/Y. Repeat the above process until beam is centered at both spot size 3 and 9 (it is OK if the beam is slightly away from the center for other spot sizes). Leave spot size at 3 (normal TEM imaging). Click “Done” to exit. Note: you must use track ball at spot size 9 and MF at spot size 3. Otherwise it won’t work. Or Spot 9 with beam shift and Spot 3 with Gun Shift.

Status window

TEM Alignment : 6 – Direct Alignment (Pivot Point)

Purpose – make sure that the beam does not shift when it is tilted.

Procedure – Condense beam to a spot (Intensity). Center using track ball. Increase Mag to 125K. Center C2 aperture and correct condenser aastigmatism if needed. Condense beam. Select “Beam Tilt pp X” to activate. Using MF X/Y knobs to make the two beams to merge to one point. Click “Done” to exit.

Beam Tilt pp Y - Mag 125K. Repeat the above step for Y direction. Click “Done” to exit.

Misalignment of beam tilt PP

TEM Alignment : 7 – Focusing Binoculars

Purpose – the binoculars provide additional magnification so that fine features can be better viewed.

Procedure - Turn the outer knob of Beam Block towards you and insert the Beam Stop from Park position. Bring in Focusing Screen. Look at the Beam Stop on the Focusing Screen through the binoculars. Focus the Beam Block by adjusting the two focusing knobs on the binoculars. Attention: - not the Focus knob on RHP!

After finishing, return the Beam Block to park position by retracting and then turning the outer knob of the Beam Stop away from you. Lower the Focusing Screen.

Beam Block

Focusing knobs

Binoculars

Focusing screen lever

TEM Alignment : 8 – Direct Alignment (Rotation Center & Beam Shift)

Purpose – make sure that the beam is along the optical axis of the objective lens.

Beam shift - Mag 125K. Condense beam to a spot. Click “Beam Shift” to activate. If the beam is away from screen center, center it using MF X/Y. If the beam becomes invisible, reduce magnification to find the beam and then bring it to screen center by using MF X/Y. Click “Done” to exit.

Rotation Center – Mag 175K or above. Spread beam to cover screen. Correct condenser astigmatism if needed. Find a sharp feature and move it to screen center using joystick. Lift up Focusing Screen and watch the feature at screen center through binoculars. Click “Rotation Center”. Minimize the image shift of the central feature using the two “Multifunction knobs”. The image should pop up like “heart beat” but does not shift. For samples with no apparent sharp features, condense beam to a point, center the beam and minimize beam shift using MF X/Y Click “Done” to exit.

Note - It is good practice to check Rotation Center regularly before taking a high resolution picture, especially when you have moved to another location on the specimen or have changed focus value more than 1 μm.

Take TEM Image Using CCD Camera

Choose the area of interest and move it to screen center.

Focus specimen and spread beam. Never view a strong condensed beam using CCD.

You may use BM-Ultrascan to take regular TEM images. Click “Search” to start viewing samples on CCD.

“Search” with binning 4 and integration 0.07-0.1 sec.

“Preview” with binning 2 and 0.5-0.7 integration.

“Acquire” with binning 1 and integration 1-2 sec.

Save image to .emi format. Right click on the image and

export data as image format (.tiff, .jpeg, etc.)

Save and Transfer Files

First, “Save As” images in .emi format (original data) to the “Images on Fei-e1390631eac” under “data” folder. If you don’t have a folder in the above folder, create one there. Then go to the Support PC. Find the “Images” shortcut on desktop, located your saved files, and copy them to the “Userdata” folder (right next to “Image” shortcut). The latter is a network storage supported by CVM IT. You can download your files from any computer on campus. After you have saved the file in .emi format, you may right click the image and “Export data”. Choose a format “w/scale marker (full resolution)”. By doing this, you can save the image in a .tiff or .jpeg format. TIA can be installed on individuals’ PC upon request so .emi files can be read directly and manipulated.

If you have not previously used the UserData folder, please ask any staff member to help set up your own folder. To retrieve the files from your office PC, also ask staff for the instructions.

If after you set it up but find you have no access to the AMCL folder, contact lab manager.

If you take images using Digital Micrograph, go to “File” menu, “Save as” image in .dm3 format and “Save display as” in image formats. Then save and transfer files in the same way.

Correct Objective Stigmation

There are two ways to correct Objective Stigmation.

1. Caustic curve – Focus image. Condense beam to a spot. Reduce “Z” so that a halo forms around the central bright spot. Go to “Tune”, click on “Objective” from the Stigmator menu. ” Adjust “MF x/y” to make the boundary of the halo round. This method is easy to perform but less accurate than the following FFT method.

2. Fast Fourier Transform (FFT) – Magnification 125k or above. Find and center an amorphous area. Acquire a live view in slow CCD (see next section for imaging with CCD camera). Click “Live FFT” in Camera menu to obtain a FFT image. Click on “Objective” from the Stigmator menu. ” Adjust “MF x/y” to make the FFT round.

Astigmatic Stigmation corrected

Focusing in TEM

Before focusing: (i) make sure eucentric height has been correctly set up. (ii) click “Eucentric Focus” on RHP. (iii) make sure C2 aperture is properly centered.

Besides making features sharp, there are three ways to assist focusing.

1) Minimum Contrast. (recommended) For all magnification. Look at image carefully. Focusing is achieved when the image has minimum contrast.

2) Fresnel Fringe. For all magnifications, look for particles, hole or edge area of the sample. Sample is out of focus if there is white (under-focus) or dark (over-focus) fringe around the edge. Adjust focus to minimize the fringe.

3) FFT. Preferred for high magnifications (>125k). Click “Live FFT” in Camera menu to obtain FFT image. Correct Obj. Stigmation. Turn Focus knob to maximize the diameter of the inner circle of FFT.

In-focus; minimum contrast

Note – use “Z-height” for rough focus and “Focus” knob for fine focus.

Out-of-focus; strong contrast

Figs. (a) under focus with objective astigmatism; (b) FFT for (a); (c) in focus with objective astigmatism; (d) FFT for (c); (e) over focus with objective astigmatism; (f) FFT for (e); (g) in focus without objective astigmatism; (h) FFT for (g).

Example of Focus and Astigmatism

High Resolution TEM

Note: HRTEM generally requires sample thickness of less than 50 nm.

Retract Objective Aperture if inserted before.

Carefully align the scope as described in previous slides.

For crystalline bulk material, accurately tilt the sample

into a major zone axis.

Locate an amorphous area. For crystalline specimen, move

to the edge of the specimen and find some amorphous

area at the edge.

Adjust the illumination so that it is in between the inner

circle and the markers on the phosphorus screen.

Lift up screen. “Preview” using CCD.

Focus sample and correct Objective Astigmatism with FFT.

(For expert user) conduct coma-free alignment in “Direct

Alignment” menu.

Acquire HREM image.

Illumination

TEM Diffraction Mode

Caution – never view a strong direct beam of a diffraction pattern using CCD without Beam Stop.

CBED (Convergent Beam)

Choose the area of interest.

Converge beam to a spot, click “Diffraction”

button on RHP. View pattern on screen.

Focus diffraction pattern with “Focus” knob.

SAED (Selected Area)

Choose the area of interest.

Insert SAD aperture by turning the lever from right to left.

Click “Diffraction” button on RHP.

Spread the beam with “intensity” knob until the pattern contains spots.

Focus diffraction pattern with “Focus” knob.

Continue on next slide …

Selected area aperture

Beam Block

TEM Diffraction Mode (Cont’d)

SAED (Cont’d)

If there is a strong central direct beam, insert the “Beam Block” and block the direct beam.

Lift up the phosphorus screen. Use Digital Micrograph. Insert CCD.

“Search” and “Start View” with 0.01 sec exposure time. Increase exposure if the pattern is dim. Stop “Search” and directly save the image.

Note:-

In Digital Micrograph, “Save as” .dm3 format and “Save display as” image formats.

If the direct beam spot is not at screen center, use “MF X /Y” to center it. If it is not circular, adjust “Stigmator-Diffraction” to make it round.

Use “Magnification” to change camera length.

The maximum tilt angle is 35° for A and 25° for B without Obj. aperture.

Camera menu in Digital Micrograph

STEM Alignment

Note: For good STEM imaging, it is important that the specimen is clean and column vacuum is below 10.

Carefully align the scope in imaging mode. Make sure Objective aperture and SAD aperture are out.

Go to “FEG Registers”, select a suitable STEM register. For HR- STEM, use spot size 9. For analysis, use spot size 3. For both, use spot size 6.

Recall a corresponding STEM alignment in “Alignment” flap out. Wait for one minute to stabilize beam.

The scope should be in diffraction mode. Otherwise click “Diffraction” on RHP.

Find an amorphous area on the sample, adjust camera length to 130 mm. Watch Ronchigram with binoculars.

Adjust “Z” to achieve focus in Ronchigram (next slide).

Move condenser aperture and center it around the Ronchigram.

Adjust “Condenser Stigmator” to make the Ronchigram round (next slide).

Move diffraction disk to center position (next slide) Click

“Insert detectors” to insert detector. Lift up screen. Use

“Search” and “Preview” to watch STEM image.

Focus image using “Focus” on RHP. Correct astigmatism using

“Condenser Stigmator”. Acquire image. Click “Auto C/B” to correct bad contrast

The circle is condenser aperture

Figure: example of Ronchigram (Au nanoparticles on carbon film). (a) overfocus; (b) in focus; (c) under-focus. (a) and (c) show shadow image of the illuminated region on the specimen. (b) Focus is achieved when no feature can be seen from inside the Ronchigram.

Phosphorus screen

inner circle

Figure: from left to right – Ronchigram with Condenser Astigmatism; Ronchigram without Condenser Astigmatism but C2 Aperture is not centered around it; Well-centered C2 aperture and no Condense Astigmatism; schematic diagram for correct position of STEM diffraction disk at 30mm camera length.

STEM Alignment (Advanced)

1. Push the “Diffraction” button on RHP to change STEM from diffraction mode to Imaging mode.

2. Use “trackball” to move beam to screen center.

3. Set magnification to 175kX or above.

4. Use “Focus” knob on RHP to condense beam to a minimum.

5. In “Direct Alignments”, select “Beam tilt ppX”. Use “MF X/Y” to make two spots merge to one. Repeat on “Beam tilt pp Y”.

6. Select “Rotation Center”, Use “MF X/Y” to minimize overall movement of the beam (like a heartbeat).

7. Select “Beam Shift” and move beam to screen center again.

8. Click “Done” to finish direct alignment.

9. Push “Diffraction” on RHP, back to Diffraction Mode.

10. Increase “Camera Length” to 100 or 150 mm. Watch Ronchigram.

11. Focus Ronchigram using “Z-height”. Re-center condenser aperture around the Ronchigram.

12. Lower camera length to 30mm. Move diffraction disk to center position.

13. Repeat 5-9 once for further tuning. Pay particular attention to #6.

14. “Search”, “Preview” image, focus with “Focus” knob and “Acquire”.

EDX - General

EDX can be performed in either TEM or STEM mode. STEM mode is preferred because of accurate positioning and much less spurious X-rays.

Mount sample to the low-background double-tilt holder. Find a thin specimen area.

Remove Objective Aperture if inserted.

Go to “INCA”, select “Option” and open “Detector Control”. Open “Shutter”.

Click “View” to observe a spectrum in TIA software. Click “Acquire’ to collect a spectrum.

Check the “Dead Time”. If it is too high (from green to red), try to reduce it by using a smaller beam size or aperture or move sample to a thinner region.

For automatic peak identification, click .

For manual peak identification, go to analysis mode .

Use the marker tool to select a peak in the EDX spectrum. Select Object Properties in the Menu View to get a list of all chemical elements matching that peak. Manually label the peaks in the spectrum by selecting/unselecting the element in the marker tab of the periodic table .

.

”

EDX in STEM Mode

Switch to STEM mode. Conduct STEM alignment.

Open “Shutter”.

Acquire a STEM image of the area of interest.

Use the position , line , or rectangular marker tool in TIA to select a spot, line, or area of interest for EDX analysis.

Set EDX acquisition time (typical 1 sec for view and 15-100 sec for acquisition) in the EDX Control Panel.

Select the position marker and view the EDX spectrum.

If the dead time appears red (above 40-50%), try reducing beam intensity (probe size, aperture).

Acquire a spectrum. Automatically or manually identify the spectrum (previous slide).

Quantification – see next slide for illustration

• Go to analysis mode

• In “View” menu, open “Periodic Table .

• Go to “ROI” in “Periodic table”. Select elements for quantification. Right click an element to choose which peak enters analysis (i.e. K, L or M)

• Click the spectrum for analysis.

• In “EDX Quant” side bar menu, click “Quantify”.

• The quantification result will appear in the message window below.

Switch to analysis mode

Right click

Spectrum quantified

Message window

TIA Analysis Window

EDX – Maps and Profiles

Acquire a STEM image as described in previous slides. See next slide for an example.

Go to “EDX” tab, at “Experiment”, select “Spectrum Collection” from “Select component” menu.

Select “Drift corrected spectrum image” for mapping or “Drift corrected spectrum profile” for line scan.

Click “Add markers” to add the spectrum position marker (orange) and drift correction marker (yellow).

Open the flap-out of “Experiment”.

Set desired image size or profile size. Set “Dwell time”. Usually 2000 for mapping, and 5000 for line scan.

Check the following options - “Acquire EDX spectra” = “Yes”; “Acquire STEM images” = “Yes”; “Post Close Col Valves” = “Yes”. The last item means that the column valve will be automatically closed once mapping or line profile is completed.

Scroll down the flat-out menu and choose the number of acquisition in slice for drift correction. Example – 15 or 20 for mapping and 5 for line scan. Press “Acquire” to start.

Configuration settings Correction settings

Spectrum position marker

Drift correction marker

Acquire a STEM image

Set up window

Setting up a drift-corrected spectrum image

EFTEM Alignment: 1 – Direct Alignment Go to “FEG Registers”, select a suitable EFTEM register. C2 aperture is placed on #4 150 μm. Both objective aperture and SAD aperture are out. Hit “Eucentric Height” and center C2 aperture. Find sample and put on eucentric height by adjusting Z. Record the sample position on “Seach” tab. Must pay close attention on Gun shift! The switch between spot 3 and 9 is crucial. This step makes sure spot table is aligned well (not shift much from spot 1 to 11) Go from spot size 1 to 11, and correct for condenser astigmatism for all spot sizes. Find a hole on the grid, where there is no carbon film or sample. Record the position of the hole on “Search tab” and type in “vacuum” underneath and click “ ” If there is no hole, close column valve and retract sample to parking position (ask staff for help). Go to “EFTEM” tab, click “EFTEM” button under “Filter” section. Look at viewing screen, the beam should condense to a smaller spot. Decrease “Intensity” turning the knob clockwise until C2 current is around 70%.

Go to “Digital Micrograph” on the right monitor. In the main menu click “Camera” – “Camera” – “EF-CCD”. Click “Start view” and lift up the screen. Look for a blank image. Simply place the mouse on the active image, and read the “Image Status” on the left column. If the “value” is a few thousand, that means we have a normal image. Click “Stop view” Go to “Camera” – “Remove Dark Reference”, and then click “Prepare Gain Reference”. In the popup window, type in target intensity 6000, Frames to average 4. Click OK – OK – Yes. Prepare Gain reference will then start automatically. “Adjust Intensity” window may pop up, if so, simply change the intensity to the green area (from red) by increasing the spot size to higher number (ex. 3 to 6). Click “Proceed”. Repeat this if more window is popped up (increase spot size to even higher). Do NOT touch any knob on the control panel or move the viewing screen when “Prepare Gain Reference” is in progress. When it’s done, put down the viewing screen. Do NOT leave the screen up unattended.

EFTEM Alignment: 2 – Prepare Gain reference

Very importantly, try to sit very still while tuning and imaging are in progress, if sitting on a

Find a workable sample area and suitable magnification. Record the position. If necessary, double check the sample on the camera. Stay at the same mag, go to the hole area, change C2 current to around 70% and spot size 3. Lift up viewing screen. Find Autofilter Panel on the right side of Digital Micrograph. Click “ ” and “Full Tune ”. Notice the buttons on AutoFilter panel turn gray symbolizing the system is busy. Wait for the automatic tuning to end. Note the result window will show progress on the tuning. Once the AutoFilter panel buttons go back to normal color, the tuning has finished. Flip down the viewing screen, go to EFTEM tab on MUI, under “Aperture” drag down menu, choose “Imaging”.

EFTEM Alignment: 3 – Gatan Image Filter (GIF) Tuning

Go back to pre-recorded sample location. Refocus if needed (go out of EFTEM mode to do so). At AutoFilter tab in Digital Micrograph, check “Slit In”, and change the Slit width to 20 eV. Click Start view on Camera. Now we should have an image. Click Align ZLP button Click to obtain a filtered Zero Loss image. A window will pop up with changeable slit width, exposure time, binning and sum frames. Change to the suitable value and click Acquire. The image will be named “Elastic” by default. Save image and rename. Click to obtain an unfiltered image. A similar window will pop up with changeable exposure time, binning and sum frames. Change to the suitable value and click Acquire. The image will be named “Unfiltered” by default. Save image and rename. Align ZLP needs to be redone periodically. If mag needs to be changed, retune the GIF by click and choose “Quick Tune”.

EFTEM Acquisition: 1 – Filtered and Unfiltered Images

EFTEM Acquisition: 2 – Jump Ratio Jump Ratio needs very stable sample so wait until sample drift stops or slow down before acquiring this. If magnification needs to be changed, conduct “Quick Tune” at the target mag first.

Click Align ZLP Click to obtain a Jump Ratio. A window will pop up with changeable pre-edge, post-edge, slit width, exposure time, binning and sum frames. Change to the suitable value and click Acquire. Click “Select Edge” to choose the interested element and edge. Click Acquire to start. It usually takes about 5-10 minutes depending on the sum frames #. The result three images include Element Pre-edge, Element Post-edge, and Element Jump-ratio. With different element, the pre-edge and post-edge energy can be adjusted. Usually silt width use 20eV. The more sum frames, the smoother the final jump ratio is. However, it is prone to suffer from sample drift as the acquisition time will be longer.

Make sure the viewing screen is down. On AutoFilter window, click “EELS”. Change spot size to 11, either on “Beam Setting” section on MUI or using the preset shortcut button on control panel. Change “Exposure” to 0.01 s. Change Aperture size to 2.5mm. Click “View”, and lift up viewing screen. If the spectrum turns red, quickly flip down viewing screen or click Stop! Now you can see a peak which is the zero loss peak, with a 2D EELS Spectrum View window below it, which shows the image of the aperture.

EELS Alignment: 1 – Align Zero Loss Peak (ZLP)

Continue on next page

EELS Alignment: 1 – Align Zero Loss Peak (ZLP)

Go to Filter Control software. Watch the aperture image. Double click FOCUS X, FOCUS Y, SX and SY, a popped up window shows the value, and move the mouse left and right could change the setting (the aperture image will change following the mouse motion). If you want to cancel the action and go back to the original value, click “ESC” to get out and back. If the alignment is satisfactory, left click the mouse to accept the change. Below shows a well-aligned filter (the first one on the left) and misaligned ones (all the others) Click Align ZLP to align the ZL peak to 0 eV. This is important b/c otherwise the energy location of acquired peak will be off. Click Stop on AutoFilter panel in DM (digital micrograph).

EELS Acquisition: Spectrum

Go to the AutoFilter in DM. Look up on energy loss peak location of specific element. For instance, B has the strong peak at 188 eV. Type in 150 eV in “Energy” box. Change exposure to 0.5 s. Click “View” to look at the spectrum. The spot size should still be 11 right now. Slowly lower it to increase the beam intensity, until spot size 1 is reached or the highest count of spectrum reach 60,000. Click “Acquire”, a window will pop up. Change the sum frames to 500~1000 depending on the count rate. The more counts there is, the more smooth and accurate the spectrum is.

Finishing Procedure

Click “Col. Valves Closed” button (color changes to yellow) to close column valves. Always close the column valve before inserting/removing the specimen holder!!

Switch back to TEM-Imaging mode (if you are using STEM or Diffraction).

Go to “Search”, click “Holder” button to reset the holder’s xyz and tilt angles to zero. (This is very important step. Failure to do so, the sample holder could be damaged during taking out).

Ask staff to help take out the holder.

Fill in the log sheet (time, the vacuum values and the emission value).