Center For Electron Microscopy and Nanofabrication

FEI Tecnai F-20 Operations Manual

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FEI Tecnai F-20 Operations Manual Last Revised: June 23rd, 2010

This manual is to be used as a guide to the basic operation of the Tecnai F-20 microscope. It should compliment the certification course offered by the Center for Electron Microscopy and

Nanofabrication (CEMN). Additional resources should be consulted for more advanced operation procedures.

                             

     

Aaron  I.  Besser  and  Zhiqiang  (Tony)  Chen  cemn.physics@gmail.com  Portland  State  University  

Center  for  Electron  Microscopy  and  Nanofabrication  

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Contents 1 Microscope Operation

1.1 FEI Tecnai F-20 TEM Anatomy 1.1.1 External Controls 1.1.2 Tecnai Optics 1.1.3 Microscope Features

1.2 Initial Conditions 1.2.1 Facility Online Management (FOM) 1.2.2 Software 1.2.3 Log Sheet & Microscope Status 1.2.4 Cold finger

1.3 Sample Preparation and Insertion 1.3.1 Single Tilt 1.3.2 Double Tilt 1.3.3 Holder Insertion 1.3.4 Microscope operation

1.4 Shutdown Procedure 1.4.1 Holder Extraction 1.4.2 Cryo Cycle 1.4.3 Software Shutdown

2 TEM Alignment 2.1 Condenser Apertures 2.2 Condenser Astigmatism 2.3 Gun Tilt 2.4 Gun Shift 2.5 Beam Pivot Points 2.6 Beam Shift 2.7 Eucentric Height 2.8 Rotation Center 2.9 Objective Astigmatism 2.10 Alignments from a File

3 TEM Analysis 3.1 Objective Focus 3.2 Image Acquisition 3.3 Selected Area Electron Diffraction (SAED)

3.3.1 Diffraction Astigmatism 3.4 Zone Axis 3.5 Bright-Field (BF) 3.6 Dark-Field (DF) 3.7 High Resolution Imaging (HRTEM)

4 STEM Alignments 4.1 Rotation Center 4.2 Pivot Points 4.3 Condenser Aperture & Condenser Astigmatism 4.4 Detector Alignment

4.4.1 Bright-Field 4.4.2 Dark-Field

5 STEM Analysis 5.1 Image Acquisition 5.2 Energy Dispersive Spectroscopy

5.2.1 Spectrum Acquisition 5.2.2 Line & Map Scans 5.2.3 EDS Quantification

6 Gatan Image Filter 6.1 Introduction 6.2 Alignment

6.2.1 Tune 6.2.2 Spectrum Alignment

6.3 Electron Energy Loss Spectrum 6.3.1 Imaging Mode 6.3.2 Diffraction Mode

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6.3.3 STEM Mode 6.4 Energy Filtered Imaging

6.4.1 Filtered Image Acquisition 6.4.2 Elemental Mapping

6.5 Spectrum Analysis 6.5.1 Background Subtraction 6.5.2 Deconvolution

7 Appendix 7.1 Alignment Quick Reference 7.2 Optimal Operating Conditions 7.3 Lens Functions 7.4 EELS Edge Reference 7.5 Characteristic X-ray Reference 7.6 Glossary

7.6.1 List of Acronyms 7.6.2 Microscope Controls 7.6.3 Terminology

7.7 References

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Chapter 1 Microscope Operation

1.1 FEI Tecnai F-20 Anatomy 1.1.1 External Controls

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1.1.2 Tecnai Optics

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1.1.3 Microscope Features • Super-twin Objective Lens • Point Resolution: 2.4Å • Line Resolution: 1.8Å • Schottky type field emission source • BF & ADF STEM • EDAX Energy Dispersive Spectroscopy • Gatan Image Filter • Single Tilt Holder • Double Tilt Holder • Gatan CT3500TR single tilt/rotate cryotransfer specimen holder • Low voltage operation

1.2 Initial Conditions 1.2.1 Facility Online Manager Users can schedule time on the microscope through the online facility management software (FOM) at https://cemn.psu.ds.pdx.edu/fom. New users must first create an account then request training through the online software. 1.2.2 Software There are four main pieces of software that must be running during microscope operation.

1. Filter Control. 2. Gatan DigitalMicrograph. 3. Tecnai imaging and analysis. 4. Microscope control.

These pieces of software must be started in the order listed above. The “application Launcher” executable will start up all of this software automatically. When logging off the machine the software must be shut down in the reverse order it was started. 1.2.3 Log sheet & Microscope Status Sign into the log sheet. If this is the first session of the day record the systems vacuum levels along with the gun information. Take note of any problems with the microscope reported by the previous users. If applicable fill out the billing information form. If you notice any problems with the machine report them to the CEMN staff via cemn.physics@gmail.com or call 503-725-9712. 1.2.4 Cold Finger Purpose: The cold finger is used to improve the vacuum conditions within the microscope and reduce contamination on the specimen. If the cold finger gets warm the vacuum may break. Procedure:

1. Fill the liquid nitrogen (LN2) canister. Cover the control pads with the plastic sheet to protect them if the nitrogen boils over. If this is the first time in the day fill the canister half way and then slowly submerge the copper coils into the canister then fill the canister the rest of the way once the boiling settles down.

2. Place the styrofoam cap on top of the canister. 3. Check the canister regularly and top off the liquid nitrogen. Do not let the level fall below the halfway point. 4. If you are the first user the LN2 will have to be filled more frequently.

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1.3 Sample Preparation and Insertion Specimens prepared for TEM imaging are placed on a 3mm grid which is held by special holders. There are three holders available to users of the Tecnai microscope. For use of the cryo transfer holder see the respective manual. 1.3.1 Single tilt holder The most basic holder. Do not touch the holder above the rubber O-ring. Do not place magnetic samples into the single tilt holder. Procedure:

1. Inspect the holder under the optical microscope for damage or debris.

a. If damaged report to facility manager. b. If dirty gently clean with Kimwipe.

2. Gently open the latch mechanism using the small pin tool stored in the plastic cover.

3. Place the grid into the specimen cup on the holder. If the specimen does not fall into the specimen cup gently tap the plastic part of the holder until the grid falls into place. Do not use tweezers to move the grid since it might damage the holder.

4. Using the pin close the latch onto the grid. 5. Flip the holder over and tap the back end to make sure that the grid is secured. The holder is now ready to be loaded into the microscope.

1.3.2 Double tilt holder This holder enables a second beta tilt axis, useful for complete control over the specimen orientation. There are three pieces used in the double tilt holder; the nut, washer, and wrench. Be careful not lose any of these components. Do not touch the holder above the rubber O-ring. Procedure:

1. Inspect the holder under the optical microscope for damage or debris.

a. If damaged report to facility manager. b. If dirty gently clean with Kimwipe.

2. Place the grid in the holder. 3. Dump the beryllium washer onto a Kimwipe, do not let it roll

away. 4. Orient the washer’s two notches so that they line up with the

notches on the holder, these should be at the one o’clock and seven o’clock positions. Gently tap the other side of the holder until the washer falls into place.

a. Check to make sure that the washer is lined up properly using the optical microscope.

5. Dump the nut onto the Kimwipe. Orient the nut such that the thinner side is down.

6. Lock the nut onto the wrench and gently place the nut on top of the holder.

7. Gently screw the nut in clockwise making sure to keep the wrench perpendicular to the holder.

a. Do not over-tighten the nut. 8. Inspect the nut under the optical microscope to ensure that it is evenly screwed in.

The holder is now ready to be loaded into the microscope.

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1.3.3 Holder insertion The holder is supported by the compustage ganiometer which controls specimen position in the x,y,z, and α directions. Procedure:

1. Make sure that the column valves are closed, the Col. Valves Closed button will be lit in yellow under the Vacuum panel in microscope control. This can also be confirmed by looking in the vacuum overview tab and making sure that the V7 valve is closed.

2. Make sure that the stage is at its home position. The X,Y,Z, α and β positions will all be around zero. a. To bring the stage to the home position press the Reset Holder button under the Stage2 expanded

tab. 3. Extract the holder from the protective plastic casing. Do not touch the holder above the O-ring. 4. Inspect the rubber O-ring.

a. If dirty ask for assistance from the facility manager. 5. Line up the pin on the holder to the closed line on the goniometer. 6. Carefully insert the holder straight into the column. Very little force is required to complete this task. 7. There is a notch on the top side of the double tilt holder, line this up with the screw at the two o’clock position.

a. The single tilt holder does not have a notch as a guide but it must still be pushed in that extra bit for proper insertion.

8. At this point microscope control will ask for the type of holder that is being inserted. Follow the instructions. a. For the double tilt holder the cable must be connected to the jack on the right side of the goniometer.

9. Wait for the turbo pump to pump down the air lock. This will take approximately 5 minutes total. 10. Once the red light turns off rotate the holder anti-clockwise until it reaches the stop.

a. If the light has been off for more than a few minutes, take out the holder and reinsert it. 11. Gently let the vacuum pull the holder into the column.

a. Hold the holder as it is pulled inwards so that it does not slam into the goniometer. 12. At this point it is safe to turn off the turbo pump by clicking the Turbo on button under the vacuum panel.

1.3.4 Microscope Operation The following conditions listed below should be met before microscope operation:

• Off button should be lit. • HT (high tension) & Vacuum buttons should be lit. • Vacuum levels:

a. Gun: 1 Log. b. Column: <15 Log.

Procedure:

1. Wait until the column vacuum recovers after the insertion of the holder. The column should be less than 15 log before beginning operation.

2. Click the Col. Valves Closed button under the Vacuum panel in microscope control. This will expose the specimen to the beam.

a. The FEG must operate in ultra high vacuum conditions, opening the column valve when the column pressure is too high will result in damage.

3. The electron beam may not appear on the screen for any of the following reasons: a. Reduce the magnification. b. Move the specimen. c. Remove the objective and SAD apertures.

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1.4 Microscope Shutdown 1.4.1 Holder extraction Procedure:

1. Make sure that the column valves are closed, the Col. Valves Closed button will be lit in yellow under the Vacuum panel in microscope control. This can also be confirmed by looking in the vacuum overview panel and making sure that the V7 valve is closed.

2. Make sure that the stage is at its home position. The X,Y,Z, α and β positions will all be around zero.

a. To bring the stage to the home position press the Reset Holder button under the Stage2 expanded tab.

3. If applicable remove data cable from compustage.

4. Grasp the holder with both hands and gently pull straight out.

a. Do not let go of the holder, it will slam into the compustage from the force of the vacuum. 5. Once the holder can’t be pulled out any further rotate it clockwise until it reaches the stopper. 6. Grasp the shaft of the holder with both hands and push the holder out with thumbs.

a. Some force is required to complete this task. Using both thumbs on the shaft provides the optimal amount of control.

7. Take the holder over to the prep table and insert into the protective plastic cover. 8. Examine the holder for any damage under the optical microscope. 9. Remove the grid and cover the holder with the plastic cap.

1.4.2 Cryo Cycle Purpose: The trapped gases on the cold finger must be released during the end of the day to ensure good vacuum conditions within the microscope. If there are no other users on the microscope for the rest of the day this procedure should be run. Procedure:

1. Under the expanded Vacuum tab press the cryo cycle button. 2. Cover the key board and microscope controls with the plastic sheet to protect the equipment from LN2 spills. 3. Remove the cold finger dewar and empty its contents into the sink in room 21.

a. Leave the dewar upside down on the workbench for the duration of the cycle. 4. After the cryo cycle delay phase is finished (~5 minutes) it is safe to close down the software and logoff.

a. In the vacuum overview it says “Cryo Cycle Running” when it has actually begun. 1.4.3 Software Shutdown At the end of session the user must shutdown the software.

1. Close the software in the opposite order which it was started. Close in the order listed below: a. Microscope control (you may save the changes) b. Technai imaging and analysis c. Gatan DigitalMicrograph d. Filter Control (do not save the changes)

2. Log off your user account. 3. Do not shutdown the computer.

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Chapter 2 TEM Alignment

2.1 Condenser Aperture Purpose: This procedure will center the condenser aperture down the optical axis. The proper alignment of this aperture has two important aspects. Convenience, changing strength of condenser lens does not change beam position. Reproducible illuminating conditions, misalignment results in beam tilt and thus changes rotation center. Center C2 Procedure:

1. Find a vacuum region. 2. Set magnification to 13500X. 3. Insert condenser aperture #4 and remove all other apertures.

a. Starting at the largest condenser aperture makes the gun tilt alignment easier. b. To use a different condenser aperture finish the gun tilt alignment then repeat this procedure with the

desired aperture. 4. Focus beam around center pip on the screen using intensity knob. 5. Center beam using beam shift track ball. 6. Expand the beam (clockwise) just around larger pip on the screen. 7. Center the expanded beam with the knobs on the condenser aperture housing. 8. Repeat steps 4 through 7 until the beam expands evenly when adjusting the intensity knob.

C1 Procedure (Only for aperture numbers 1, 2 and 3): The C1 aperture is used for EDS, EELS, and other advanced data collection. For most cases the C1 aperture will not be used and it should be set to position #4.

1. Reduce the magnification. 2. Spread the beam to a maximum with the intensity knob. 3. Center the aperture in the screen with the aperture housing knobs.

Notes:

• It is very important to align this aperture in a reproducible manner so that the rotation center does not have to be changed every time the aperture is changed.

• The alignment of the condenser aperture will change between STEM (nanoprobe) and normal (microprobe) imaging modes. When entering a new mode the aperture should be realigned.

2.2 Condenser Astigmatism Purpose: To ensure that the illuminating beam is circular. This allows for easier alignments of other components later. Condenser astigmatism results from some irregularity in the levels of focus present in the condenser lenses. Procedure:

1. Find a vacuum region. 2. Set

a. Magnification to 125 kX or above. b. Spot size 2.

3. Click the condenser button under the stigmator panel. 4. Condense the beam with intensity knob (counter-clockwise). 5. Adjust the multifunction x and y knobs to make the beam completely circular.

Notes:

• Each multifunction knob controls a perpendicular direction in the stigmator coils. • It often helps to adjust the condenser lens strength (intensity knob) through a range of values to correct the

astigmatism. • The condenser astigmatism will be different for each spot size and condenser aperture.

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2.3 Gun Tilt Purpose: Important for having maximum beam intensity. Adjusts the gun deflection coils so that the electron beam is emitted parallel to the optical axis. This ensures that all the electrons produced can be used for imaging. Procedure:

1. Find a vacuum region. 2. Set

a. Magnification at 125 kX or above b. Spot size 2 c. Obj. Lens at ~91.8%

3. Click gun tilt under the direct alignments panel. 4. Condense the beam with the intensity knob until the gun image

can be seen. a. The gun image will appear as a central bright spot in the

center of the illuminating beam (when well aligned). 5. Set the sensitivity of the multifunction knobs to a minimum

(press the minus button several times). 6. Carefully adjust the gun tilt (multifunction knobs) until the central

bright spot is in the center of the illuminating beam. a. It may be necessary to adjust condenser lens astigmatism during the course of this alignment.

Notes:

• After correcting gun tilt it may be necessary to correct gun shift. • If the gun tilt is far off the central bright spot may not be seen making it more difficult to correct. • It is easier to do this alignment with the largest condenser aperture, #4.

2.4 Gun Shift Purpose: Adjust the gun deflection coils such that the electron beam travels straight down the optical axis through the center of the C1 lens. Changing spot size will not cause beam to shift. Procedure:

1. Find a vacuum region. 2. Set

a. Magnification at 125 kX b. Obj. Lens at ~91.8%

3. Click gun shift in the direct alignments panel. 4. Change spot size to 9. 5. Move beam to center of screen with beam shift trackball. 6. Reduce spot size to 3. 7. Move beam to center of the screen using gun shift (multifunction)

knobs. 8. Repeat steps 4 through 7 until beam shift is minimized between

spot sizes. Notes:

• After correcting gun shift it may be necessary to correct gun tilt again. It may be nessessary to pe • Changing spot sizes will change the condenser astigmatism, it is not necessary to correct for this during the

course of this alignment.

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2.5 Beam Pivot Points Purpose: To decouple the beam tilt and beam shift operations, meaning that beam tilt will not occur when the beam is shifted and vise versa. Important for high resolution imaging in which a beam tilt would result in a change in rotation center, for scanning in STEM mode where distortions would be caused by a changing beam tilt. Procedure:

1. Find a vacuum region. 2. Set

a. Magnification to 125 kX. b. Spot size 2.

3. Condense beam too slightly larger than inner pip using intensity knob. 4. Set the multifunction knobs to correct pivot point x by selecting beam tilt ppx from the Direct Alignments

panel. 5. Converge the two bright spots using the multifunction knobs. 6. Set the multifunction knobs to correct pivot point y by selection beam tilt ppy from the Direct Alignments

panel. 7. Converge the two bright spots using the multifunction knobs.

Notes:

• There are two pivot point adjustments which can be made for each direction. The compensator will have to be adjusted more (multifunction x) than the corrector (multifunction y).

2.6 Beam Shift Purpose: To ensure that the beam entering the objective system is parallel to the optical axis. This will minimize aberrations in the final image. Procedure:

1. Click beam shift under the Direct Alignments panel. 2. Move the beam to the center of the screen using the multifunction knobs.

a) The trackball cannot be used because it is not a pure beam shift.

2.7 Eucentric Height Purpose: Important for minimizing specimen shift when tilting, proper alignments later, and more accurate magnifications. Method 1: diffraction method

1. Set Obj current value to ~91.8% with focus knob. Or press the eucentric focus button. 2. Remove objective aperture. 3. Over an amorphous region (like lacy carbon) condense the beam to a point using the intensity knob. 4. Minimize airy rings by adjusting the specimen height (Z-axis) buttons.

Method 2: alpha wobbler

1. Set Obj current value to ~91.8% with focus knob. Or press the eucentric focus button. 2. Engage alpha wobbler under the expanded stage2 tab. 3. Minimize image movement by adjusting the specimen height (Z-axis) buttons.

Method 3: eucentric focus

1. Press the eucentric focus button. 2. Adjust specimen height (Z-axis) buttons until the image is in focus, minimum contrast.

Notes:

• The first method is useful when determining Gaussian defocus is difficult.

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• The second procedure allows for a more subjective interpretation. • The third method is only useful when it is easy to determine the Gaussian defocus condition.

2.8 Rotation Center Purpose: To ensure that the beam is along the optical axis of the objective lens. Such that changes in the objective lens strength do not shift the image. Procedure:

1. Find a recognizable area of the specimen. 2. Set

a. Bring the specimen to in-focus, Obj Lens ~91.8%. b. Magnification 125 kX.

3. Expand the beam with the intensity knob (clockwise) off the screen. 4. Under the Direct Alignments panel select Rotation center to set multifunction knobs. 5. Minimize image movement with the multifunction knobs.

a. Use the lift up screen and binoculars for finer adjustments. Notes:

• First try minimizing movement in a direction with one multifunction knob then minimize movement in the other direction.

• During this process the beam may shift off the screen, expand it to a point in which this does not occur. This will occur more if the pivot points are not set properly.

2.9 Objective Astigmatism Purpose: To correct the non-uniformity in the focal length of the objective lens by setting the stigmators to compensate for this distortion. Method 1: Without camera

1. Find a planar hole in the specimen. 2. Set magnification to 125 kX. 3. Set multifunction knobs to objective stigmators under the stigmator panel. 4. Bring the image to slightly underfocus using the focus knob (counter-clockwise).

a. Bright Fresnel fringes will become visible inside the hole. 5. Balance the Fresnel fringes by adjusting the objective stigmator (multifunction knobs).

Method 2: With camera

1. Find a region of amorphous material (like lacy carbon). 2. Set magnification to 125 kX. 3. Click objective under the stigmators panel. 4. Bring the specimen to slightly under-focus using the focus knob (counter-clockwise). 5. Expand the beam until it is far off the screen using the intensity knob (clockwise). This is very important for

preventing damage to the CCD. 6. Navigate to the CCD/TV Camera panel in microscope control. 7. Under the Camera drop down menu select CCD and click insert. 8. Lift the screen by pressing either R1 or the Screen lift button to the left of the column. 9. Click search and locate the amorphous material on the screen. 10. Take the FFT (Fast Fourier Transform) of the image by going to Process → Live → FFT menu option. 11. Make the concentric rings as circular as possible by adjusting the objective stigmator (multifunction knobs).

Notes:

• The first technique is more difficult since it allows for a more subjective interpretation of the image. It may also be difficult to locate a planar hole in the specimen depending on the type of grid used. For these reasons it is often preferable to use the CCD camera.

• It can also help to draw a circle on the screen to help align this pattern, under the Standard tools window select the circle and hold down the shift key to draw a perfect circle around the FFT.

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• The rings will be hard to see if the specimen is completely in focus, try moving to slightly underfocus.

Figure 1 - Fourier transforms of amorphous carbon with various degrees of objective astigmatism.

2.10 Alignments from a File Purpose: Convenience, a rough alignment of the column can be obtained by loading a previous set of optical conditions stored in a file. This reduces the amount of time spent on alignments in many cases. Procedure: Alignment File

1. In the expanded alignments panel click the file tab. There is a list of current files available.

2. Select the desired file 3. Under the available section double click all of the desired alignments you

wish to apply to the machine. 4. Click Apply.

Procedure: FEG Register

1. On the expanded FEG Registers panel click open. 2. Open the appropriate .feg file containing the gun settings desired.

a. In the image below the customer service file is opened. 3. Under the FEG Registers panel select the desired alignment file

depending on the optical conditions desired. Notes:

• FEG Registers can be especially useful for switching back and forth between STEM and normal imaging modes.

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Chapter 3 TEM Analysis

3.1 Objective Focus Focus is a measurement of how far the image plane is from the specimen plane. When the image plane is above the specimen the resulting image is said to be “over-focused” (a more strongly excited objective lens). When the image plane is below the specimen then the resulting image is “under-focused” (A weakly excited objective lens). When the image plane is in the same position as the specimen plane then the image is “in-focus” or at Gaussian defocus (Objective lens = 91.8%). Defocus is measured in units of length and is usually on the order of nanometers and is controlled by the focus knob on the right control pad. In some cases it may be difficult to determine the current focus condition. The current focus condition manifests in the final image as changes in the fresnel fringe around the edges of the specimen and in the frequency domain as changes in the radius of the airy rings, see figure 2. Over-focused images will have a dark fresnel fringe and airy rings of finite radius in the frequency domain. Under-focused images will have a bright fresnel fringe and airy rings of finite radius in the frequency domain. In-focus images will have no fresnel fringe and airy rings of infinite radius in the frequency domain. There are a few important focus values for certain types of analysis. For normal imaging it is best to operate the image slightly under-focused, Obj. Lens <91.8%. For high resolution imaging the spatial frequencies present in the image are dependant upon the specific focus condition. At Scherzer defocus the number of spatial frequencies will be maximized. This occurs at approximately -60nm.

Figure 2 - Series of images showing different focus conditions and how they manifest.

3.2 Image Acquisition For normal TEM imaging the gatan CCD camera will be used to acquire all the images. This camera sits below the viewing screen in the column. Images acquired are managed by the DigitalMicrograph software and by default are saved in the proprietary .dm3 file format. Images can be exported to different data types but this may cause the loss of useful metadata (like scale information). If the Gatan software is not available the free java based software called ImageJ can be used to open .dm3 files instead. It can be downloaded from: http://rsbweb.nih.gov/ij/. Procedure: Basic Image Acquisition

1. Align the microscope in TEM mode. 2. Find region of interest. 3. Reduce the beam intensity by expanding the beam with the intensity knob, clockwise. 4. Lift the screen by pressing either R1 or the Screen lift button to the left of the column.

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5. Click the search button in microscope control. 6. Immediately hover the mouse pointer over the brighter regions of the image. In the Image Status window note

the pixel “Value”. If this value is above 8,000 units reduce the brightness by turning the intensity knob clockwise (expanding the beam or reducing the exposure time) OR lower the screen.

a. The camera scintillator can be damaged if the beam is too intense. This can be especially true when imaging diffraction patterns.

b. Limiting the camera’s exposure to the beam will increase its lifespan. c. There are diminishing returns for intensities above a few thousand, so there is no reason to increase

intensity beyond this value. 7. To capture an image press the Acquire button.

a. The amount of exposure time can be adjusted by clicking the up and down arrows to the right of Integration time.

Notes:

• Do not change the magnification when the screen is up. • Always check and reduce the brightness before lifting the screen. • Minimize exposure of the camera to the beam this will increase its lifespan. • Use the beam stop to block the forward scattered spot when capturing SAED patterns.

3.3 Selected Area Electron Diffraction (SAED) When entering diffraction mode (by pressing the diffraction button) the microscope changes the projection lens current so that the back focal plane is projected onto the screen or detector. A diffraction pattern is a picture of the distribution of scattered electrons exiting the sample. There are several different types of diffraction patterns that can be produced depending on the imaging conditions and the specimen properties. Procedure: Selected Area Diffraction

1. Align the microscope in TEM mode. 2. Tilt the specimen to the desired Zone axis. See Zone axis procedure. 3. Insert selected area aperture and center it with the aperture housing knobs.

a. This aperture controls which areas of the specimen will contribute to the diffraction pattern. b. Turn the toggle switch on the bottom to the left to insert the aperture. c. Larger apertures will produce a more intense diffraction pattern.

4. Center the area of interest in the aperture. 5. Press the diffraction button on the right control pad. 6. Expand the beam to the maximum with the intensity knob, clockwise. 7. Focus the diffraction pattern with the focus knob. 8. Block the forward scattered beam with the beam stop by twisting the knob counter-clockwise until it drops into

the column. a. To remove the needle twist the knob clockwise while gently pulling out.

9. To capture the pattern on the CCD: a. Make sure the intensity of the pattern is low as possible and the beam block is in. b. Decrease the integration time in the search mode to as low as it will

go. c. Raise the screen. d. Press the search button. e. IMMEDIATELY check the intensity of the image at the brightest

points. f. LOWER the screen if the pixel value is above 8,000.

Procedure: Diffraction Astigmatism

1. Remove SAD and Objective apertures. 2. Expand the beam clockwise to the maximum, with the intensity knob. 3. Press the diffraction button. 4. Adjust the focus of the pattern until the caustic image is visible.

a. It may be necessary to increase camera length significantly with the magnification knob.

b. If the image is still hard to see move the specimen such that the beam is in the center of one of the grids.

5. Click diffraction under the stigmators panel.

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6. Adjust the astigmatism with the multifunction knobs until the pattern forms a three pointed star. Notes:

• The diffracted beams can be extremely bright making it difficult to capture them on the CCD camera without causing damage. This actually makes it impossible to capture some types of patterns on the camera. The use of the beam stop is important along with increasing the strength of C2 to dim the beam. If the pattern is still too bright try decreasing the size of the SA aperture or the condenser aperture.

• The camera introduces a magnification so before capturing the image decrease the camera length. The camera is not exactly down the optical axis so it might be necessary to shift the diffraction pattern slightly to the left.

• Assuming that the specimen is at eucentric height it can be tilted with the α and β to tweak which zone axis it is on.

3.4 Zone Axis The zone axis is a direction along the intersection of two or more crystallographic planes. Diffraction patterns are dependant upon the orientation of the crystal with respect to the incident beam hence making finding the right zone axis important. The goniometer on the microscope is capable of α tilt, with the addition of the double tilt holder a β tilt is also possible. These two tilts combined allow the user to select any desired zone axis in the crystal. Method 1: Kikuchi Pattern (For Thick Specimens) Kikuchi patterns enable the accurate determination of crystal orientation. They allow the user to easily get a sense of tilt since the pattern behaves as if it were fixed to the bottom of the crystal. This pattern results from multiple scattering events and hence thicker specimens may be required. The intersection of the bands within the pattern represent a particular zone axis.

1. Align the microscope in TEM mode. 2. Enter nanoprobe mode by clicking nanoprobe in the beam settings panel. 3. Focus the beam to a spot over the crystal with either the intensity knob or by clicking focus beam on the beam

settings panel. 4. Press the diffraction button to project the Kikuchi pattern onto the screen. 5. Tilt the specimen along one of the “roads” until the zeroth-order spot lands on one of the band intersections

corresponding to the desired zone axis. Method 2: Tilt with BF image

1. Form a bright field image with the smallest objective aperture, #4. a. See the bright field imaging procedure.

2. Tilt the specimen along the dark band inside the crystal until specimen has darkest contrast. Method 3: Tilt with SAED pattern

1. Bring specimen to the eucentric height. 2. Insert SA aperture over the region of interest. 3. Press the diffraction button to enter diffraction mode. 4. Tilt the specimen such that three diffraction spots are continuously visible until the pattern becomes

symmetric about the forward scattered spot. a. Note that the diffraction pattern is simply a projection of the intersection of the Ewald sphere with the

reciprocal lattice. Once zone axis is reached the reciprocal lattice will be symmetric around the Ewald sphere.

Notes:

• Method 1 is generally the easiest but requires thick specimens. • Method 2 can be used to achieve a rough alignment first before continuing on to method 3.

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3.5 Bright Field Imaging When all of the diffracted beams are excluded from the final image using the objective aperture the result will be a bright field image. Dark regions in this image will correspond to areas which are diffracting beams and bright regions will correspond to areas which are not diffracting beams. This imaging method will enhance diffraction contrast. Procedure:

1. Align the microscope in TEM mode. 2. Illuminate an area of the specimen that is diffracting beams (Amorphous carbon will work). 3. Enter diffraction mode by pressing the diffraction button. 4. Center the forward scattered spot on the screen with the multifunction knobs, diffraction shift. 5. Insert the smallest objective aperture, #4. 6. Center the aperture on the forward scattered spot. 7. Press the diffraction button again to go back into normal imaging mode.

Notes:

• The objective aperture is used to select which beams will contribute to the final image. In bright field the diffracted beams are excluded which results in enhanced contrast, for this reason the objective aperture is sometimes referred to as the contrast aperture.

• It may be necessary to correct for objective astigmatism again upon the insertion of an objective aperture.

3.6 Dark Field Imaging Dark field imaging occurs when the objective aperture is used to select a diffracted beam to form the final image. This imaging mode has inverted contrast compared to bright field imaging. Bright regions will correspond to areas which are diffracting beams at the particular angle selected. Centered Dark Field:

1. Find the desired zone axis and enter diffraction mode. 2. Press the dark-field button. 3. Use the multifunction knobs to center the desired diffracted spot in the center pip.

a. One knob controls the radius and the other controls the angle of deflection in the coils. 4. Insert the smallest objective aperture (10µm, #5) around the diffraction spot. 5. Exit diffraction mode. 6. Pressing the dark field button will switch between a bright field image and a dark field image.

3.7 High Resolution Imaging This imaging mode of the microscope allows the examination on the atomic scale. Contrast results from differences in the phase of electron waves exiting the specimen. Similar to SAED this method can be used to determine crystallographic structure. Procedure:

1. Align the microscope in TEM mode. a. HRTEM imaging is extremely sensitive to operating conditions within the microscope. b. Image quality is proportional to the time spend on alignments.

2. Place the plastic cover over the compustage to isolate the holder from vibrations. 3. Find the desired zone axis. 4. Insert the proper objective aperture to select diffracted and transmitted beams. 5. Increase magnification until lattice fringes can be seen.

a. It may not be possible to see the lattice fringes on the main screen, use the lift up screen and binoculars.

b. It may be necessary to correct for objective astigmatism. 6. Project the image onto the CCD camera and view it on the computer.

a. See image acquisition procedure. 7. Adjust the focus until scherzer defocus is reached.

a. Looking at the image in the frequency domain may help to identify the frequencies. Notes:

• Scherzer defocus occurs around -60nm for the Tecnai microscope operating at 200 kV.

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λsCf 2.1−=Δ Approximation of scherzer defocus value.

• The choice of the objective aperture depends on several factors. • Specimen drift may become an issue for larger exposure times. • The Gatan software comes with a set of tools for easy masking to enhance particular spatial frequencies.

Chapter 4 STEM Alignments Scanning Transmission Electron Microscopy is an operating mode available in the Tecnai F-20 instrument. In this mode images are formed by rastering a focused beam across the specimen then collecting the electrons that emerge to form an image. In this way the function of a STEM is very similar to a SEM. Due to the change in operating conditions when entering STEM mode the microscope will have to be realigned in nanoprobe mode.

4.1 Rotation Center Purpose: To ensure that the beam is along the optical axis of the objective lens. Minimize lens aberrations and image movement during focusing. Procedure:

1. Exit diffraction mode by pressing the diffraction button. 2. Select rotation center under the Direct Alignments panel. 3. Center the central bright spot using the multifunction knobs.

a. It may be necessary to adjust the focus to see this bright spot. Notes:

• The focus step knob will change the amplitude of the wobble during the rotation center alignment.

4.2 Pivot Points Purpose: Proper alignment of the pivot points in STEM mode is essential to eliminate image distortions and ensure more accurate magnification. This process will decouple the beam tilt and shift. Procedure:

1. Enter imaging mode. 2. Click beam tilt ppx under the Direct Alignments Panel. 3. Converge the two bright spots with the multifunction knobs. 4. Click beam tilt ppy 5. Converge the two bright spots with the multifunction knobs.

4.3 Condenser Aperture & Astigmatism Ronchigram Also referred to as the Gabor hologram or central zero-order disc of the convergent beam electron diffraction pattern. Looking at this pattern provides the best way of precisely aligning the microscope in STEM (nanoprobe) mode. This technique will enable the alignment of the condenser aperture, the condenser astigmatism, and will allow the user to bring the image to in-focus. It is a useful picture showing the manifestation of spherical aberrations in the electron lenses.

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Figure 3 - Computer generated Ronchigram by J M Rodenburg.[ref###]

Procedure: Generate Ronchigram

1. Enter STEM mode. 2. Enter Diffraction mode. 3. Set camera length to 150mm. 4. Insert the largest condenser aperture, #4. 5. Move the specimen to an amorphous region. 6. Raise the lift up screen with the lever on the right hand side of the column. 7. Shift the pattern with the diffraction shift coils, multifunction knobs. 8. View the pattern through binoculars.

Procedure: STEM Condenser Astigmatism

1. Click condenser in the stigmators panel. 2. Insert the largest condenser aperture, #4. 3. View the Ronchigram through the binoculars. 4. Make the Distortion rings around the outside edge of the pattern as circular as possible, with multifunction

knobs. Procedure: STEM Condenser Aperture

1. Select the desired condenser aperture by turning the aperture housing knob. 2. Using the aperture housing knobs adjust the aperture position such that it is even with the Ronchigram.

4.4 Detector alignment 4.4.1 Bright field detector Procedure:

1. Remove the Gatan Ultrascan CCD camera by clicking insert under the camera panel. 2. Insert the STEM bright field detector by depressing the button on the control box. 3. Set the camera length above 100mm. 4. Using the diffraction shift move the beam to the 9 o’clock position around the inner pip on the phosphorous

screen. 5. Raise the screen and click the search button under the STEM imaging panel. 6. Reduce the magnification and maximize the brightness of the image using the diffraction shift.

4.4.2 Annular dark field detector This detector generates an image from the diffracted electrons exiting the specimen. In addition to diffraction contrast Z contrast is also possible. Procedure:

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1. Reduce the camera length to 40mm or lower.

a. A higher camera length allows for Z-contrast imaging. 2. Align the beam on STEM bright field detector, see above procedure. 3. Insert the STEM dark field detector by depressing the button on the control box.

a. The dark field detector is positioned around the BF detector. Notes:

• It is possible to use the TV camera in the GIF system to align this detector but some care must be taken to not damage the GIF CCD.

Chapter 5 STEM Analysis

5.1 Image Acquisition Procedure:

1. Find the area of interest while using the continuous search. 2. Focus the image using the focus knob. 3. Set the desired contrast and brightness levels either

manually under the STEM detector panel or automatically by clicking the Auto C/B button.

4. To acquire an image click acquire under the STEM imaging panel.

a. Dwell time and resolution can be adjusted under the STEM imaging panel.

5.2 Energy dispersive spectroscopy The Tecnai is equipped with an EDAX EDS system with ultra-thin window. This instrument is capable of examining the characteristic x-rays emitted from the sample for elements as low as carbon. 5.2.1 – Spectrum acquisition Procedure:

1. Bring the sample to ~15 degrees alpha tilt. This will maximize the EDS signal. 2. Locate the area of interest and acquire an image. 3. Select the dispersion desired by going to the drop down box in the expanded EDX panel.

a. This will determine the energy window and how long the probe dwells. 4. Select the type of scan desired (point, line, or area) from the toolbox to the right of the TIA

software and place it on the image. a. The line and area scans will raster the beam in the selected area.

5. Click acquire to begin collecting the spectrum. a. To end collection prematurely click acquire again.

Notes:

• The optimal dead times are between 20-30%. Dead times falling outside of this range will result in artifact peaks.

• To increase dead times increase the current to the sample by: reducing the spot size, increasing condenser aperture size, reducing the gun lens, or increasing the extractor. Dead times can be reduced by doing the opposite.

5.2.2 – EDS Line and Map Scan Procedure:

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1. Acquire a STEM image. 2. Under the Experiments panel: Select component → SpectrumCollection 3. Under the Experiments panel: Select experiment

a. Drift corrected spectrum profile for a line scan b. Drift corrected spectrum image for a map scan.

4. Place the box over the region for drift correction. A larger box will increase the acquisition time. 5. Set up the scan parameters in the expanded panel.

a. Acquisition Settings i. Profile size or Image size controls the number of points along the line or direction. ii. Dwell time controls the acquisition time for each point.

b. Configuration Settings i. Acquire EDS Spectra: Yes ii. Acquire STEM Images: Yes iii. Elemental Process: No

c. Correction Settings i. Number of acquisitions in slice: controls the number of EDS profiles to be taken between

looking at the drift correction image. 6. Highlight the other line or box and position it over the region of interest. 7. Press the acquire button to begin the data collection.

Analysis

8. Open up TIA by clicking the blue icon on the bottom right of microscope control.

9. Select the image position marker tool and click onto the line or image. Hold down the alt key, click and drag the marker tool onto the spectrum.

a. You must drag onto the actual spectrum not just the graph. b. Drag the marker tool around the line or image to view the spectrum at that point.

10. To generate an elemental profile: a. Identify the peaks either manually or using the automatic peak identification by clicking the peak ID

button under EDX Quant tab. b. Select the energy window tool and draw a window around peak. c. Under the Unary tab click extract map/profile near the bottom.

Notes:

• Alternatively the Automap function can be used which will identify elements and generate maps automatically. 5.2.3 – EDS Quantification Procedure:

1. Collect spectrum with a dead time of ~20-30%. a. Optimal dead times are extremely important for accurate quantification.

2. Go to the TIA window by clicking the blue icon in the bottom right of microscope control. 3. Under the Quant Setup tab click Spectrum Conditions. 4. Set the thickness and mass density of the specimen. 5. Under the EDX Quant tab click Quantify.

a. The quantification information will appear in the analysis window near the bottom. Notes:

• Precise quantification is very difficult and requires a detailed understanding of the sample and machine parameters.

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Chapter 6 Gatan Image Filter

6.1 Introduction When the electron beam passes through the specimen it losses energy through a variety of different processes. These inelastically scattered electrons contain additional information about the specimen. The GIF system is used to sort out and collect this information. The energy distribution of this signal contains a large amount of information about the elemental composition and valence states of the sample. This technique is considered complimentary to EDS since it has improved detection of lighter elements and a higher energy resolution. The GIF system consists of several different components located on the base of the column, see figure ###. Electrons are passed through a magnetic prism which sorts them out based upon their energy. A series of magnetic lenses project the resulting signal through an energy slit and then onto the GIF CCD camera. Images are captured using the Gatan DigitalMicrograph and the Filter Control software. The GIF system is extremely sensitive to external interference. Small changes in the surrounding electromagnetic fields will cause large changes in the alignment of the GIF system. For this reason it is recommended to use to the wooden chairs (which lack movable magnetic components). There is a field cancellation system around the room which should eliminate some interference.

6.2 Alignment The GIF must be aligned precisely in order for it to function. Any change in the conditions within the microscope will require it to be realigned. A smaller condenser aperture will improve the spherical and chromatic aberrations but at the cost of brightness. 6.2.1 Tune This procedure must be preformed at the beginning of the session and every time the magnification or other optical conditions are changed. The GIF system comes with a set of scripts which will perform most of the alignments automatically. Procedure:

1. Align the microscope in TEM mode. 2. Set the desired magnification.

a. The prism introduces an additional 20X magnification. 3. Go to a vacuum region. 4. Set the GIF to imaging mode, the imaging button will be lit. 5. Turn on the TV monitor. 6. Click TV image in the AutoFilter panel. 7. Set the GIF to imaging mode in the AutoFilter panel, the imaging button will

be lit. 8. Center the beam at the 9 o’clock position of the center pip. 9. Expand the beam to the edge of the screen with the intensity knob,

clockwise. 10. Raise the screen and view the image on the TV monitor. 11. Make sure the image is not too bright.

a. Reduce intensity if it is. 12. Extract the TV camera by clicking the TV image button in the AutoFilter

panel. 13. Click Align ZLP. This process will take 1-2 minutes.

a. Follow the directions given by the software. It may ask you to adjust the intensity, do so with the intensity knob.

b. Repeat this step if the energy shift is more than 2eV. 14. Click the Tune GIF button in the AutoFilter panel. This process will take 3-5

minutes. a. Follow the directions given by the software. It may ask you to adjust

intensity. b. Repeat this step.

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6.2.2 Spectrum Alignment This procedure will allow the user to adjust the lenses within the spectrometer to optimize the data collection. This alignment should be done immediately after having done the Tune alignment. Procedure:

1. Lower the phosphorous screen. 2. Click Spectroscopy in the AutoFilter panel. 3. Expand the beam beyond the screen and confirm this by clicking enter in the TIA box. 4. Select entrance aperture: Mask. 5. Reduce the search time to a minimum. 6. Click search in the AutoFilter panel.

a. The spectrum will be displayed in DigitalMicrograph and the ZLP should be visible. b. If the spectrum is yellow or red IMMEDIATELY lower the phosphorous screen and decrease the

intensity. 7. Adjust intensity such that the height of the spectrum is ~1500 units above background.

These next alignments are preformed using the Filter Control software in the Adjustments window. Double click the alignment then drag the mouse left or right to adjust the value. The scroll wheel adjusts the sensitivity of the mouse movement. Pressing escape will reset the value to its previous state.

8. Focus X & Y adjustment.

a. Maximize the height of the ZLP for each of the directions. 9. SX & SY concavity adjustment.

a. Maximize the height and minimize the width of the ZLP for each of the directions. 10. ACComp A & B cancellation field adjustment.

a. Reduce the drift of the ZLP for each direction. 11. Use energy shift to move the ZLP to zero on the energy axis.

Notes:

• It is possible to do these alignments by looking at the spectrum on the TV camera.

6.3 Electron Energy Loss Spectrum This section covers the different methods of spectrum acquisition. Before acquiring the spectrum align the GIF system. Do not change the magnification without redoing GIF alignment. 6.3.1 Imaging Mode Procedure:

1. Lower phosphorous screen and center the feature at the 9 o’clock position at the center pip. 2. Decrease the intensity of the beam with the intensity knob. 3. Click spectroscopy in AutoFilter. 4. Click enter to confirm the reduction in intensity. 5. Select the desired entrance aperture.

a. 0.6mm or 2mm apertures are often the best. b. A smaller entrance aperture will improve the energy resolution at the cost of intensity.

6. Reduce the search time to a minimum (0.01s). 7. Select the region of interest in the spectra by either clicking the Zero Loss, Plasmon, Pre-C, or Custom

button. a. If a custom energy shift is desired enter the appropriate value in the energy shift section then click the

enter button. 8. Raise the phosphorus screen. 9. Click search.

a. Adjust the intensity such that the signal is visible. As the energy loss increases the signal decreases so it may be necessary to increase the intensity.

b. Note the search time and highest intensity of the signal on the graph. 10. Adjust the acquisition time such that the GIF CCD is not over exposed. 11. Click Acquire

Notes:

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• When the microscope is in imaging mode the GIF system is called diffraction-coupled. • If the spectrum ever becomes yellow or red IMMEDIATELY lower the screen.

6.3.2 Diffraction Mode Procedure:

1. Click spectroscopy in the AutoFilter panel. 2. Insert the TV camera by click the TV Image button in the AutoFilter panel. 3. Enter diffraction mode by pressing the diffraction button. 4. Center the forward scattered spot at the 9 o’clock position around the inner pip using diffraction shift,

multifunction knobs. 5. Set the desired energy shift such that the edges of interest are visible.

a. Do not project the ZLP onto the GIF CCD while in diffraction mode. 6. Raise the screen. 7. View the spectra on the TV monitor.

a. If the spectrum is too intense lower the screen and decrease the intensity. 8. Retract the TV camera by clicking the TV Image button. 9. Set the search time to a minimum (0.01s). 10. Click search.

a. The spectrum should be visible in DigitalMicrograph. 11. Note the intensity of the spectrum on the search and adjust the acquire time accordingly. 12. Acquire the spectra.

Notes:

• When the microscope is in imaging mode the GIF system is called image-coupled. • The Tune GIF and Align ZLP routines cannot be preformed while the microscope is in diffraction mode. • If the spectrum ever becomes yellow or red IMMEDIATELY lower the screen.

6.3.3 STEM Mode The microscope can be configured to simultaneously obtain an EELS spectra while generating an ADF STEM image. This allows the user to generate EELS data from small volumes while correlating it to an image. Procedure:

1. Align the GIF system while in nanoprobe imaging mode. a. See procedures from section 6.2

2. Set the desired energy shift such that the edges of interest are visible. a. Be careful when projecting the ZLP onto the GIF while in STEM mode as it may be to bright

3. Insert the TV camera by clicking the TV Image button in the AutoFilter panel. a. This will block the beam from hitting the GIF CCD.

4. Enter STEM mode. 5. Perform the STEM alignments described in chapter 4. 6. Align the beam onto the ADF detector. See procedure 4.4.2 7. Acquire a STEM image. 8. Dwell the beam at a position of interest using the beam marker tool. 9. Look at the spectra on the TV camera and make sure that it is not too intense. 10. Set the search time to a minimum (0.01s). 11. Extract the TV camera. 12. Click search.

a. The spectrum should be visible in DigitalMicrograph. b. At this point the beam position can be moved by dragging the red point around on the acquired STEM

image. 13. Note the intensity of the spectrum on the search and adjust the acquire time accordingly. 14. Acquire the spectra.

Notes: • If the spectrum ever becomes yellow or red IMMEDIATELY lower the screen. • Current must be controlled precisely for this technique to work. There is a delicate balance between intensity

of the spectra and STEM spatial resolution. It is always better to start with less current (higher spot size, smaller C2) and work up to higher currents.

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6.4 Energy Filtered Imaging Energy filtered imaging allows the user to generate images using electrons with specific energies. This technique has the advantage of very high spatial and energy resolutions. 6.4.1 Filtered Image Acquisition Procedure:

1. Enter EFTEM, Imaging, & Filtered mode by clicking the buttons in the AutoFilter panel. 2. Enter desired energy shift corresponding to the region of interest. 3. Enter the desired energy slit value.

a. This will select the range of electron energies which will be used to generate the image. 4. Center the image on the entrance aperture by moving it with the beam shift trackball to the 9 o’clock position

at the inner pip. 5. Under the camera panel select the GIF CCD and click search.

a. The GIF system introduces an additional magnification. 6. Bring the image to in-focus with the focus knob. 7. Check the intensity by hovering the mouse over the image.

a. If it is over 8000 lower the screen and adjust the intensity. 8. Adjust the exposure time, binning, and entrance aperture such that the pixel values are around 4000.

a. Adjust the exposure time by adjusting the integration time in the CCD/TV panel. b. Adjust the binning by navigating to the expanded tab on the CCD/TV panel, select acquire from the

drop down box at the lower right, and change the binning. c. Adjust the entrance aperture by going to the AutoFilter panel and selecting a new aperture from the

drop down menu. d. Note that these settings will be different for the search, preview, and acquire modes of image capture.

Notes:

• The higher the energy shift the lower the intensity of the image. 6.4.2 Elemental Mapping The GIF system can automatically generate elemental maps. This allows the user to generate images whose contrast contains elemental information. This technique yields similar results to EDS maps but with much higher spatial resolutions. Three different EFTEM images are acquired: two pre-edge and one post-edge. Subtraction routines are then used to generate an image where contrast corresponds to electrons of that particular energy loss. Procedure:

1. Enter EFTEM, Imaging, & Filtered mode by clicking the buttons in the AutoFilter panel. 2. Acquire an energy filtered image of the region of interest and note the acquisition parameters required to

achieve the desired intensity (~4000). see section 6.4.1

3. Click the El. Map button on the lower part of the AutoFilter panel.

a. A new dialog window will be opened. 4. In the Elemental Mapping Setup select the desired

parameters. a. To select an element type in the atomic

number in the element box. b. Select an edge by clicking the edge

button. c. Set the slit width. Use the same value

determined in step 2. d. Select the position of the slits for each

image acquisition. Two pre-edge and one post-edge.

e. Additional options are available in the mapping options submenu. 5. Click OK when complete. 6. Set the exposure in the next dialog box. Use the same value determined during step 2.

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7. Align the resulting images by dragging the images with the mouse while holding down the Ctrl key until the image movement is minimized.

Notes:

• Longer exposure times may be required. Be sure not the damage the GIF CCD. • Remember to use the wooden chair. • To change the parameter in the Elemental Mapping Setup page click other items.

6.5 Spectrum Analysis The Gatan DigitalMicrograph software comes with several different functions that are useful for analyzing spectra. The options listed below are available on the microscope computer. Additional plugins are available online. 6.5.1 Background Subtraction Edges of interest are often small compared to the background signal. It is easier to interpret the spectrum after this background has been removed. Procedure:

1. Hold down the Ctrl key, click and drag across the pre-edge spectrum. 2. In the EELS menu → Background Model → Extract Background Subtracted Signal

a. The resulting spectra will be displayed in another window. Notes:

• The background model can be changed by going to the EELS menu → Background Model is → Preferences. 6.5.2 Deconvolution The resulting spectra can be modeled as a convolution between the plural scattering and edge spectra functions. To isolate the edge spectra a deconvolution routine can be preformed. In theory, this method can be used to completely remove the plural scattering but in practice it is effectively limited to lower-loss spectra. Two different methods are described below. Method 1: Fourier-log Used on low-loss spectra which contain the ZLP.

1. Bring the desired low-loss spectrum to the front. 2. Navigate to EELS → Select Remove Plural Scattering → Fourier-Log.

a. The resulting spectra will appear in a new window. Method 2: Fourier-ratio Used on higher-loss spectra where the ZLP is not visible.

1. Acquire a low-loss spectrum. 2. Acquire an edge spectrum under the same experimental conditions. 3. Remove the pre-edge background. See procedure 6.5.1 4. Navigate to EELS → Remove Plural Scattering → Fourier-ratio. 5. Select the window which contains the low-loss spectrum. 6. A new window will display the deconvoluted spectra.

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Chapter 7 Appendix

7.1 Alignment Quick reference Initial conditions

• Software: GIF, DigitalMicrograph, TIA, Microscope Control. • Fill out log sheet and billing information. • Full LN2 Dewar, Closed Col Valve, Stage at Home.

Condenser Aperture

• Magnification: 13500X • Condense beam to center pip, center with beam shift trackball; Expand beam to larger circle, adjust aperture

knobs. Condenser Astigmatism

• Magnification: 125kX, Multifunction: Condenser stigmators. • Condense beam, adjust multifunction to make beam circular.

Gun Tilt

• Magnification: 125kX; C2 aperture: #4; Multifunction: Gun Tilt, reduce sensitivity. • Condense beam, center central bright spot with multifunction.

Gun Shift

• Magnification: 125kX; Crossover beam; Direct Alignments: Gun Shift • Spot: 3; center beam with Multifunction knobs • Spot: 9; center beam with beam shift trackball • Repeat

Pivot Points

• Magnification: 125kX; Crossover beam • Multifunction: ppx, converge spots • Multifunction: ppy, converge spots

Beam Shift

• Magnification: 125kX; Crossover beam; Direct Alignments: Beam shift • Move beam to center of screen using beam shift trackball

Eucentric Height

• Magnification: 125kX; Crossover beam onto carbon. • Adjust Z-axis until no airy rings.

Rotation Center

• Magnification: 125kX; Expand beam past screen; Multifunction: Rotation Center; Specimen in focus • Minimize image movement

Objective Astigmatism

• Magnification: 125kX; Expand beam; insert CCD camera; Multifunction: Obj. Stig. • Raise screen and search for image. • Process → Live → FFT • Adjust multifunction until rings are circular.

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7.2 – Optimal operating conditions 7.2.1 – TEM Bright Field and Dark Field Imaging Mode: TEM BF (microprobe) C1: 2mm, #4 C2: Any Obj.Ap.: 10µm, #5 Gun Lens: 1-5 Spot Size: 1 Focus: Slightly under-focused, Obj. Lens < 91.8% 7.2.2 – High-Resolution Imaging Mode: TEM BF (microprobe) C1: 2mm, #4 C2: 50-100 µm, #1-3 Obj.Ap.: Depends, used to select specific range of diffracted beams. Gun Lens: 3-5 Spot Size: 1 Focus: Scherzer, Defocus = -60nm. 7.2.3 – EDS Analysis Mode: STEM (nanoprobe) C1: 30µm, #1 C2: 150µm, #4 Obj.Ap.: None, #7 Gun Lens: 5-8 (depending on current required) Spot Size: 6-9 (depending on current required) Focus: In-focus, Obj. Lens = 92.5% 7.2.4 – Selected-Area Electron Diffraction Mode: TEM BF (microprobe) C1: 2mm, #4 C2: Any Obj.Ap.: None, #7 Gun Lens: 3-5 Spot Size: Any Focus: In-focus, Obj. Lens = 91.8%

7.3 Lens & Aperture Functions High Magnification Low Magnification

Obj. lens Image focus Diffraction (LAD) focus Diff. lens Diffraction focus Image focus

Obj. aperture Contrast forming Area selection SA aperture Area selection Contrast forming

Obj. stigmator Image stigmation Diffraction stigmation Diff. stigmator Diffraction stigmation Image stigmation

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7.4 EELS Edge Reference

Element Edge Energy (eV) K L M N O

1 H 13 - - - - 2 He 22 - - - - 3 Li 55 - - - - 4 Be 111 - - - - 5 B 188 - - - - 6 C 284 - - - - 7 N 401 - - - - 8 O 532 - - - - 9 F 685 - - - - 10 Ne 867 18 - - - 11 Na 1,072 31 - - - 12 Mg 1,305 51 - - - 13 Al 1,560 73 - - - 14 Si 1,839 99 - - - 15 P 2,146 132 - - - 16 S 2,472 165 - - - 17 Cl 2,822 200 - - - 18 Ar 3,203 245 12 - - 19 K 3,607 294 18 - - 20 Ca 4,038 346 25 - - 21 Sc 4,493 402 32 - - 22 Ti 4,966 456 35 - - 23 V - 513 38 - - 24 Cr - 575 42 - - 25 Mn - 640 49 - - 26 Fe - 708 54 - - 27 Co - 779 60 - - 28 Ni - 855 68 - - 29 Cu - 931 74 - - 30 Zn - 1,020 87 - - 31 Ga - 1,115 103 - - 32 Ge - 1,217 29 - - 33 As - 1,323 41 - - 34 Se - 1,436 57 - - 35 Br - 1,550 69 - - 36 Kr - 1,675 89 11 - 37 Rb - 1,804 110 14 - 38 Sr - 1,940 133 20 - 39 Y - 2,080 157 26 - 40 Zr - 2,222 180 29 - 41 Nb - 2,371 205 34 - 42 Mo - 2,520 227 35 - 43 Tc - 2,677 253 39 - 44 Ru - 2,838 279 43 - 45 Rh - 3,004 307 48 - 46 Pd - 3,173 335 51 - 47 Ag - 3,351 367 56 - 48 Cd - 3,538 404 67 - 49 In - 3,730 443 77 - 50 Sn - 3,929 485 24 - 51 Sb - 4,131 528 31 - 52 Te - 4,341 572 40 - 53 I - 4,557 619 50 - 54 Xe - 4,782 672 63 - 55 Cs - - 726 76 - 56 Ba - - 781 90 15 57 La - - 832 99 14

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58 Ce - - 883 110 20 59 Pr - - 931 113 22 60 Nd - - 978 118 21 61 Pm - - 1,027 120 - 62 Sm - - 1,080 129 21 63 Eu - - 1,131 133 22 64 Gd - - 1,185 140 20 65 Tb - - 1,241 147 25 66 Dy - - 1,295 154 26 67 Ho - - 1,351 161 20 68 Er - - 1,409 168 29 69 Tm - - 1,468 180 32 70 Yb - - 1,528 185 24 71 Lu - - 1,588 195 28 72 Hf - - 1,662 214 31 73 Ta - - 1,735 229 36 74 W - - 1,809 245 36 75 Re - - 1,883 260 35 76 Os - - 1,960 46 45 77 Ir - - 2,040 60 50 78 Pt - - 2,122 71 52 79 Au - - 2,206 83 54 80 Hg - - 2,295 98 58 81 Tl - - 2,389 118 75 82 Pb - - 2,484 138 19 83 Bi - - 2,580 157 24 90 Th - - 3,332 335 88 92 U - - 3,552 381 96

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7.5 Characteristic X-ray Reference

Element Peak Energy (keV)

Kα Lα Mα 4 Be 0.110 - - 5 B 0.185 - - 6 C 0.282 - - 7 N 0.392 (Ti) - - 8 O 0.523 (V, Cr) - - 9 F 0.677 (Fe) - - 10 Ne 0.851 - - 11 Na 1.041 (Zn) - - 12 Mg 1.254 - - 13 Al 1.487 (Br) - - 14 Si 1.740 (Rb, Sr, Ta, W) - - 15 P 2.015 (Mo, Pb) - - 16 S 2.307 (Mo, Pb) - - 17 Cl 2.622 - - 18 Ar 2.957 - - 19 K 3.312 (Cd, In) - - 20 Ca 3.690 0.341 - 21 Sc 4.088 0.395 - 22 Ti 4.508 (Ba) 0.452 (N) - 23 V 4.949 (Ti, Cr) 0.510 (O) - 24 Cr 5.411 (V) 0.571 (O) - 25 Mn 5.895 (Cr) 0.636 - 26 Fe 6.400 (Mn) 0.704 (F) - 27 Co 6.925 0.775 - 28 Ni 7.472 0.849 - 29 Cu 8.041 0.928 - 30 Zn 8.631 1.009 (Na) - 31 Ga 9.243 1.096 - 32 Ge 9.876 1.186 - 33 As 10.532 1.282 - 34 Se 11.210 1.379 - 35 Br 11.907 1.480 (Al) - 36 Kr 12.630 1.587 - 37 Rb 13.375 1.694 (Si, Ta, W) - 38 Sr 14.142 1.806 (Si, Ta, W) - 39 Y 14.933 1.922 - 40 Zr 15.746 2.042 (P) - 41 Nb 16.584 2.166 - 42 Mo 17.443 2.293 (S, Pb) - 43 Tc 18.327 2.424 - 44 Ru 19.235 2.558 - 45 Rh 20.167 2.696 - 46 Pd 21.123 2.838 - 47 Ag 22.104 2.984 - 48 Cd 23.109 3.133 (K) - 49 In 24.139 3.287 - 50 Sn 25.193 3.444 - 51 Sb 26.274 3.605 - 52 Te 27.380 3.769 - 53 I 28.512 3.937 - 54 Xe 29.669 4.111 - 55 Cs 30.854 4.286 - 56 Ba 32.065 4.467 (Ti) - 57 La 33.302 4.651 0.833 58 Ce 34.569 4.840 0.883 59 Pr 35.864 5.034 0.929

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60 Nd 37.185 5.230 0.978 61 Pm 38.535 5.431 1.032 62 Sm 39.914 5.636 1.081 63 Eu 41.323 5.846 1.131 64 Gd 42.761 6.059 1.185 65 Tb 44.229 6.275 1.240 66 Dy 45.728 6.495 1.293 67 Ho 47.257 6.720 1.348 68 Er 48.818 6.948 1.406 69 Tm 50.410 7.181 1.462 70 Yb 52.035 7.414 1.521 71 Lu 53.693 7.654 1.581 72 Hf 55.362 7.898 1.645 73 Ta 57.106 8.145 1.710 (Si, Rb) 74 W 58.864 8.396 1.775 (Si, Sr) 75 Re 60.655 8.651 1.843 76 Os 62.482 8.910 1.910 77 Ir 64.346 9.173 1.980 78 Pt 66.246 9.441 2.051 79 Au 68.185 9.711 2.123 80 Hg 70.106 9.987 2.195 81 Tl 72.176 10.266 2.271 82 Pb 74.228 10.549 2.346 (S, Mo) 83 Bi 76.321 10.836 2.423 84 Po 78.460 11.128 2.501 85 At 80.636 11.424 2.581 86 Rn 82.855 11.724 2.662 87 Fr 85.124 12.029 2.744 88 Ra 87.437 12.338 2.825 89 Ac 89.790 12.650 2.911 90 Th 92.190 12.966 2.996 91 Pa 94.643 13.291 3.082 92 U 97.143 13.613 3.171

7.6 Glossary 7.6.1 List of Acronyms

ADF Annular Dark Field BF Bright Field CBED Convergent Beam Electron Diffraction CCD Charge Coupled Device DF Dark Field DP Diffraction Pattern EDS Energy Dispersive Spectroscopy EELS Electron Energy Loss Spectroscopy EFTEM Energy Filter Transmission Electron Microscopy FFT Fast Fourier Transform GIF Gatan Image Filter HRTEM High Resolution Transmission Electron Microscopy IGP Ion Getter Pump ROI Region Of Interest SAED Selected Area Electron Diffraction SEM Scanning Electron Microscope/Microscopy STEM Scanning Transmission Electron Microscope/Microscopy TEM Transmission Electron Microscope/Microscopy TIA Tecnai Imaging and Analysis ZLP Zero Loss Peak

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7.6.2 Microscope Controls

α Tilt Located on the left control pad. This controls the tilt in the α direction using the compustage goniometer. This function is useful for finding zone axis. β Tilt Located on the left control pad. This allows for a new tilt direction for complete control over the orientation of the specimen it is only enabled when using the double tilt holder. Beam Shift Trackball Trackball located on the left control pad which controls the beam deflection coils above the specimen. It allows the user to move where the spot is located on the specimen. Control pad There are two control pads to on both sides of the column. They each contain knobs, buttons and trackballs which control various microscope functions. Dark field The dark field button located on the right control pad allows the user to easily perform centered dark field imaging. Pressing this button changes the function of the multifunction knobs so that the user can control the diffraction deflection coils. Multifunction X controls the deflection angle and Multifunction Y controls the deflection radius. Diffraction The diffraction button located on the right control pad allows the user to project the back-focal plane (where the diffraction pattern is located) onto the screen. Eucentric focus This button is located on the right control pad and allows the user to change the objective lens current to its optimal value. Focus This knob is located on the right control pad. In normal imaging mode it controls the position of the image plane with respect to the specimen by adjusting the objective lens strength. In STEM mode it controls the position of the spot by adjusting the condenser lens strength. Intensity This knob is located on the left control pad and controls the current through the second condenser lens. Adjusting this will change the size of the spot of the screen and allows the user to control the brightness of the beam. L1 Reset defocus. L2 Alpha wobbler. L3 Reduce spot size. Magnification There are several magnification modes. For most alignments it is assumed that the microscope is in the SA range.

Mh: 440kX → 910kX. SA: 6,200X → 400kX. M: 2250X → 4400X. LM: 19X → 1850X. Multifunction There is one multifunction knob on each control pad, X and Y. The function of these knobs are set by the user through the microscope control software. To check the current function of the knobs see the lower left pane in the software. On/Off These buttons located on the control box to the right of the column allows the user to turn on/off the microscope. Do not press these buttons. R1 Screen Lift. R2 Toggle micro between nanoprobe modes. R3 Increase spot size. Screen lift The button located just to the left of the column, not to be confused with the close V7 button, will raise the screen without going through the microscope control software. Specimen Shift Trackball This trackball is located on the right control pad controls the position of the specimen using the compustage. The two buttons above the trackball control the sensitivity. STEM Detectors Located just above the right control box this panel allows the user to insert or extract the STEM detectors. Pressing the circle button will insert the BF detector and the ring button will insert the DF detector. To extract the detectors press the button to the right with the arrow Stigmator This button will change the multifunction knobs to adjust a set of stigmator coils dependant upon which operating mode the microscope is in. V7 Close This button located to the left of the column and further back on the panel will close the column valve in cases of emergency without going though the microscope control software. In cases of emergency or when the software is unresponsive and the column needs to be closed press this button. Wobbler Engages the alpha wobbler on the goniometer. Z-axis The two z-axis buttons on the right control pad control the position of the specimen in the z direction, up and down the column.

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7.6.3 Terminology Accelerating Voltage The electrical potential electrons experience after exiting the gun which causes them to accelerate. This controls the energy of each electron. Aperture A hole or opening which the electrons travel through. There position is controlled through the aperture housing knobs on the side of the column. Astigmatism Astigmatism refers to an uneven level of focus in the lens. There are three different types of astigmatism that the user can correct for: Condenser, Objective, and Diffraction. Backfocal plane The plane just below the specimen plane which contains the diffraction pattern. Beam Blanker A deflection coil which shifts the beam completely off the specimen. Beam Stop Located on the right of the column this device is used to block out the zero-order or forward scattered spot so that it does not damage the CCD chip. Binning The combination of several pixels into one. Increasing the binning reduces image size and increases intensity. Binoculars These are used for a finer inspection of the image projected onto the lift up screen. Brightness The current density per unit solid angle of the source with SI units of A·M-2·sr-1. C1 Aperture The top aperture on the column. For most cases this aperture will be left out, in position #4. This aperture is located just above the crossover below the C1 lens. C2 Aperture The second aperture from the top of the column. For most cases this aperture will be used as the beam defining aperture and is located in the second condenser lens. Center pip The small central ring on the phosphorus screen. Cold Finger Improves the vacuum conditions within the column and prevents contamination. Column The main section of the microscope that houses the optics and detectors. Column Valve Also referred to as the V7, this valve isolates the gun from the rest of the column. Compustage This devices controls the position of the specimen within the column. Condenser Lens This lens controls the size of the probe on the specimen by adjusting the

current through the second condenser lens. The user can adjust this parameter using the intensity knob in normal imaging mode. In STEM mode this lens controls the focus. Contrast The difference in intensity between two adjunct areas. Quantitatively defined as:

oIIC Δ= where C is the contrast, ΔI is the

change in intensity, Io is the reference. The human eye generally cannot perceive contrast less than 10%. Current The flow of charge per unit time. Cryo-holder Used for holding specimens at cryogenic temperatures while simultaneously imaging them. Capable of an α tilt and LN2 temperatures. Deflection Coil A coil which produces a magnetic field to tilt the beam. This tilt angle is changed by changing the current through the coil. Deflection coils come in pairs so that a pure beam shift can be preformed. There are several sets of these coils in the column. Defocus The physical distance between the image plane and the specimen plane. Controlled by the objective lens in normal mode and the condenser lens in STEM mode. Diffraction pattern The image of the distribution of electrons after exiting the specimen. Diffusion Pump A type of vacuum pump used by the Tecnai microscope. Double Tilt Holder This holder enables a second tilt axis in the β direction giving the user complete control of the orientation of the specimen. Dwell Time The amount of time the beam dwells at a particular pixel in the STEM image. Energy dispersion Refers to the energy per pixel in the electron energy loss spectra. Adjusting this value will alter the intensity of the spectra. Eucentric Height Refers to the z position of the specimen that is optimal for the microscope. Exposure Time Exposure time controls the duration in which the CCD camera is exposed the electron beam and is collecting the signal. Longer exposure time increase the intensity of the signal.

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Extractor The anode plate below the FEG tip which draws electrons out of the tungsten filament. FEG emission A measure of the amount of current from the gun to the extraction anode. This should be on the order or ~60µm. Field-cancellation system The Tecnai room is surrounded with a field cancellation system that limits the amount of electromagnetic interference. The control box is located on the right side of the work bench. Field Emission Gun The component of the microscope responsible for generating the electron beam. The Tecnai is equipped with schotkey field emission gun. Forward scattered spot Also referred to as the zeroth-order spot (000). This beam in the diffraction pattern is formed by electrons which have been not been scattered by the specimen. Gatan Image Filter This system has several different components that allow the user to form images using electrons of particular energies or spectra showing the distribution of electron energies. GIF CCD The CCD which forms both EEL spectra and EFTEM images. This camera is extremely sensitive and expensive. Goniometer The device on the column which provides an α tilt. Guassian Defocus Refers to the focus condition in which the image plane is at the specimen plane, objective lens ~91.8%. This is the same as in-focus. Gun Lens An electrostatic lens just below the gun which controls how spread of the electron beam entering the condenser system. High-tension The voltage applied to the anodes below the gun which accelerate the electrons to high energies. Holder Refers to the device which holds the TEM specimen. Image Plane The plane which is being projected onto the screen or CCD. The position of this plane is controlled by the objective lens. In-focus Refers to the focus condition in which the image plane is at the specimen plane, objective lens ~91.8%. This is the same as Guassian defocus. Intensity The amplitude of signal being generated by the detector, related to the current

per unit area (flux). High intensities will damage the CCD. Kikuchi Pattern Refers to the pattern of lines, bands, or roads that constitute a kikuchi map. These patterns are useful for orientation of the specimen. LN2 Dewar The container holding the liquid nitrogen (LN2) for the cold finger. Microprobe The normal operating mode of the microscope in which the mini-condenser lens is not activated. Nanoprobe An operating mode of the microscope mainly used for STEM. An additional condenser lens is activated to achieve a smaller probe size. Objective Aperture This aperture is the third from the top of the column. In normal imaging mode this aperture lies in the back-focal plane just below the specimen, where the diffraction pattern is located, and is used for selecting which beam is going to contribute to the final image. When in low magnification mode this aperture swaps functions with the selected area aperture. Objective Lens This lens collects and focuses the electrons that have interacted with the specimen. Optical Axis The center of the lenses, apertures, and column. Over-focus Refers to the focus condition in which the image plane is above the specimen plane, strongly excited objective lens >91.8%. Panel A section in the microscope control software, which contains a set of operations or functions. The position of the tabs will be different for each users account. To adjust the position of these tabs select workspace layout in the pop up at the lower right part of microscope control. Phosphorous screen For most imaging and alignments the beam will be projected onto this screen. Pivot Point A point around which the beam will pivot such that tilt and shift are decoupled. Projection Lenses A series of lenses which magnify and project the image onto the screen or camera. Raster Refers to the scanning that is preformed while in STEM mode. A large raster area corresponds to a lower magnification and vise versa.

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Ronchigram The hologram used for aligning the microscope in STEM (nanoprobe) mode. Scherzer Defocus Refers to the focus condition in which the spatial frequencies in the HRTEM image are maximized. This occurs at approximately -60nm for the Tecnai F-20. Scintillator The device used to amplify the signal to the CCD chip. Selected Area Aperture This is the lowest aperture on the column. In normal imaging mode it is used to select the area of the specimen which will contribute to the diffraction pattern. It lies in the conjugate image plane. In low magnification mode this aperture swaps functions with the objective aperture. Single Tilt Holder The standard holder used for holding the specimen. This holder only has a single tilt in the α direction. Specimen Plane The plane in which the TEM specimen is. Spot size Refers to the size of the probe on the specimen, higher spot size will correspond to a smaller probe size. This is controlled by adjusting the first condenser lens current. Stage Cover Placed over the compustage to isolate the holder from vibrations. STEM Detectors When the microscope is in STEM mode the resulting images are formed using these solid state detectors. Stigmator Refers to the device which corrects for the astigmatism present in a lens. There are several sets of stigmators.

Tab A user defined section under the workset which contains panels. Turbo A type of vacuum pump used by the Tecnai microscope. TV Camera This is part of the GIF system and is used a sacrificial camera to protect the GIF CCD. Images can be viewed on the TV monitor. TV monitor This monitor is hooked up to the GIF TV camera just before the GIF CCD and allows the user to view the image that is to be projected onto the GIF CCD. It is used as a sacrificial camera so that the more expensive GIF CCD is not damaged. Ultra-scan CCD The standard CCD camera used for capturing images and diffraction patterns. It is below the phosphorous screen. Under-focus Refers to the focus condition in which the image plane is below the specimen plane, weakly excited objective lens <91.8%. Vacuum Region A region on the specimen which minimally interacts with the electron beam. Valve A component in the vacuum system which blocks the flow of gases. Workset The section of microscope control which contains a set of user defined tabs.

Zone axis A specific specimen orientation in which two crystallographic planes are intersecting.

7.7 References

1. David B. Williams and C. Barry Carter. Transmission Electron Microscopy. 1996. 2. Dr. Chunfei. Operational Procesdures for Tecnai F-20 TEM. May 30th, 2003. 3. Micah C. Eastman. JEOL 100CX TEM Operations Manual. June 18th, 2008. 4. Dr. Daniel Abou-Ras. Manual for energy-dispersive x-ray measurements at the FEI TECNAI F30. February

9th, 2006. 5. Gatan, inc. DigitalMicrograph EELS Analysis User’s Guide. July 2003, revision 1.2.1 6. J. M. Rodenburg. http://www.rodenburg.org. Tutorial courses in transmission electron microscopy. Accessed

on 5.3.10. 7. FEI TECNAI on-line Help TECNAI F20-TECNAI F30. 8. FEI TECNAI 20 Operating Manual 9. FEI on-line Help Manual--TIA