TECNAI T12 September 2013 Start-of-Session Steps
1. Add LN2 to ACD dewar. Check level during session. DO NOT allow to dry!
2. Log into Windows with your username and password.
3. Open TECNAI USER INTERFACE; Open TIA.
4. Load Sample. Put sample onto chosen sample holder (single tilt; double tilt.) Observe O-ring under optical microscope and remove dirt and dust.
To insert holder into microscope:
- Align small pin on holder with large pin on goniometer and insert holder fully into microscope. Push holder in and rotate clockwise approximately 1/4 turn until holder enters ~1 cm more into microscope. Do not turn any more; push in firmly.
- Follow prompts on computer: “Select Sample Holder” – click ↵ - “Connect Cable” – click ↵
- Wait for red light to turn off.
- Turn sample holder anti-clockwise. Slowly guide rod fully into microscope. DO NOT let sample rod slide in by itself!
5. Generate Beam. On SETUP workset, find the FILAMENT window. If HIGH TENSION and FILAMENT are ON (both buttons Yellow) skip to Step 6.
- Select desired kV from pull down list. Change kV ONLY when FILAMENT is off!
- Click HIGH TENSION button (Yellow = On.) Wait for EMISSION to return to less than 1.0.
- Click FILAMENT button (Yellow = On.) Wait for Filament Heat to reach saturation (blue line meets red line.)
- Change STEP value, if desired, but DO NOT set so that EMISSION becomes >10 µA.
6. Check VACUUM. Wait for GUN/COL to be < 20 Click COL. VALVES CLOSED to open valves. (Button Yellow = Closed; Button Grey = Open)
7. Find Beam. If no beam is seen, lower magnification, move stage, change beam INTENSITY. At LM magnifications remove condenser aperture.
8. Select and align Condenser Aperture. Condense beam to cross-over with INTENSITY knob. Center beam with Track Ball. Spread beam with INTENSITY until beam is same size as TEM screen. Align Condenser Aperture so beam edge matches screen edge. (See Step 13, Objective Aperture Alignment, for image showing location of aperture alignment controls.)
9. Set Eucentric Height. Find region of interest on sample. Start ALPHA WOBBLER. Stop the image motion by pressing Z buttons. (Repeat this step whenever moving to a new region on the sample.)
10. Focus Image. Focusing image may be easier on computer monitor: INSERT camera; start SEARCH. Fine tune focus with FOCUS knob. Change focus STEP by turning bottom half of knob, but DO NOT use STEP higher than 6. Option: press WOBBLER button (above FOCUS knob) to aide in focusing – out of focus image divides into two images – merge images to focus. If focus is very far from correct, press EUCENTRIC FOCUS button to obtain near correct focus.
11. Align Beam. Do DIRECT ALIGNMENTS found on ALIGN page. (Repeat these whenever making significant changes in operating conditions, or to improve present alignment.)
GUN TILT – Spread beam to fill screen. Use MULTIFUNCTION X and Y to maximize brightness on screen.
GUN SHIFT – Condense beam to cross-over. Select SPOT SIZE 9. Center beam with the track ball. Select SPOT SIZE 3. Center beam with MF X and Y. Repeat.
BEAM TILT PP X and Y – Use MF X and Y to converge two beams and stop beam from “shaking.”
BEAM SHIFT – Use MF X and Y to center beam.
ROTATION CENTER – Spread beam to obtain image on screen. Set Magnification >59000x. Center a recognizable object on center of screen with joystick. Use MF X and Y to stop object from moving away from center of screen. Image should appear to move “up and down” the column centerline, but not move side to side.
12. Diffraction Alignment. Insert and align the Selected Area Aperture. Switch to diffraction mode by pressing DIFFRACTION button. Center diffraction pattern with MULTIFUNCTION X & Y knobs. Change “size” of diffraction pattern (i.e. Camera Length) with MAGNIFICATION knob. Insert Objective Aperture (see below for guide). If edge of aperture is unclear, use FOCUS knob to sharpen. Remove Objective Aperture. Focus the diffraction pattern with INTENSITY knob.
13. Objective Aperture Alignment. (Inserting Objective Aperture will improve image contrast.) While in Diffraction Mode, insert Objective Aperture by moving Insertion Lever to the left. Select aperture size of choice by turning Size-Selector Knob (4 is largest, 1 is smallest.) Align aperture so the bright undiffracted spot is in the center of aperture. Press DIFFRACTION button to switch back to image mode.
alignment knob alignment knob
insertion lever size-selector knob
14. Objective Stigmation. If image will not focus well at high magnification, adjust the objective stigmation: Press STIGMATOR button on left panel. Use MULTIFUNCTION X and Y to obtain well focused image.
Objective stigmation mis-adjusted Objective stigmation well adjusted
Changing Samples During Session
1. Open STAGE workset; tab-out STAGE2. Click “HOLDER” in ‘Reset’ area to set stage positions to zero.
2. Open SETUP workset. Close valves by clicking COL VALVES CLOSED. (Grey = Open; Yellow = Closed)
3. Leave HIGH TENSION and FILAMENT on.
4. Remove sample holder:
- Press 2 fingers of one hand on purple part of goniometer.
- Pull holder straight out until stop.
- Turn holder clockwise toward CLOSED until stop (do NOT pull while turning.)
- Pull holder fully straight out (do not hesitate or move holder inward while pulling out.)
- Disconnect double-tilt cable.
5. Remove sample from holder; replace with new sample.
6. Insert sample holder into microscope according to step 4 on page one.
7. Check VACUUM window. Wait for GUN/COL to be < 20
Click COL. VALVES CLOSED to open valves. (Button Yellow = Closed; Button Grey = Open)
8. Always set EUCENTRIC HEIGHT (step 9) and FOCUS (step 10) with new sample. It is not necessary to repeat DIRECT ALIGNMENTS unless needed.
Using TIA
1. General Information
TEM Imaging & Analysis (TIA) software is a rich environment with many functions that permit acquiring data (images, spectra, etc.) as well as analyzing saved data. The TIA program displays on the right-hand monitor, while Tecnai User Interface (which controls the TEM) displays on the left- hand monitor. The mouse cursor can move freely between the right-hand and left-hand monitors.
TIA has two major modes of operation, ACQUISITION mode and ANALYSIS mode. The purpose of having two operating modes is to facilitate each operation with different menus, shortcuts, and data windows.
ACQUISITION mode window appears as:
In this example, two “panes” are open showing an image and a diffraction pattern. Typically only a single, larger pane will be showing. Note the menu icons on the right panel. Placing the mouse cursor over any icon will bring up the name or function of that icon.
ANALYSIS mode window appears as:
In this example many “panes” are visible, containing images, spectrum images, and spectrum data. Note the “shortcut” tabs on the left side of the window, information tabs on the right, and more extensive menus and icons across the top and bottom.
2. Setting Up Mode Selection
To set up TIA to actively switch between ACQUISITION and ANALYSIS mode, locate the TECNAI 2 G power bar in the Tecnai User Interface window:
From the pull-down menu, select “Application Preferences.”
Find “TEM Imaging & Analysis” in the pull-down menu in the Application Preferences window:
Select the Normal button to enable switching between modes. Select either Acquire Only or Analyze Only to lock that preference. Click Apply to set changes.
3. Selecting Modes
2 To switch between ACQUISITION and ANALYSIS mode, locate the TECNAI G power bar (see above) in the Tecnai User Interface window. The blue-colored icons are the controls for switching.
In ACQUISITION mode the bar appears as:
Click the _ to switch to ANALYSIS mode.
In ANALYSIS mode the bar appears as:
Click the L to switch to ACQUISITION mode.
NOTE: WHEN IN ANALYSIS MODE, THE TECNAI UI ON THE LEFT-HAND MONITOR DISAPPEARS! DO NOT BE ALARMED BY THIS. IN ANALYSIS MODE THE UI IS NORMALLY NOT NEEDED.
ON SYSTEMS WITH ONLY ONE PC MONITOR, THE INTENTION OF HIDING THE UI IS TO GIVE MORE ROOM FOR THE ANALYSIS MODE WINDOW AND SHORTCUTS.
4. ACQUISITION Mode
In ACQUISITION mode, TIA is optimized for the collection of data: images and spectra. Any number of image or data panes may be open at one time in ACQUISITION mode, although usually only the most recently viewed one will be showing. At the bottom of the window are tabs to select each of the acquired images/data for viewing.
E.g.:
All the controls to actually acquire data are built into the Tecnai UI program on the left-hand monitor. The relevant controls can be found on CAMERA, EDX, or STEM tabs in the UI.
Many tools that increase the functionality of ACQUISITION mode are available as icons on the tool bar located on the right side of the window. Place the mouse cursor over an icon to learn its name or function.
The most commonly used icons on the right panel are:
Open Close Close All Arrange Save Sequentially Save As Auto Save
Show Info Panels Show Data Window
Shortcuts
While most of the analysis tools are available in ACQUISITION mode, it is more convenient to perform these steps while in ANALYSIS mode. Switch modes by clicking the appropriate icon on the UI power bar.
NOTE: Closing TIA while in ACQUISITION mode. There is no “close window” icon (the X in upper right corner) in ACQUISITION mode. To close (quit) the TIA program, press ALT + F4. Any unsaved images/data will be lost and cannot be recovered.
5. Saving Images
Click SAVE AS, or SAVE SQUENTIALLY (saves image with number added to name), or AUTO SAVE (image automatically saved as soon as acquired with sequential numbering) icons while selected image is showing. TIA saves images as two files with .emi and .ser extensions. When session is done, you may convert the images to .tif files following steps below.
Batch Converting Files to .tif Click SHORCUTS icon then select FOLDER EXPROT in ACQUSITION mode, or find the FOLDER EXPORT tool in left-hand column in ANALYSIS mode. Click SETTINGS to open dialog window as below.
Enter the exact location of the folder containing the files to be converted in SOURCE FOLDER line. This folder must contain ONLY .emi and .ser files, and must contain BOTH file types for each image. Other file types – Spectra, Profiles, etc.– may also be converted in the same way, but this is best done by first moving unique file types to separate folders. Enter desired location of folder to contain converted images in TARGET FOLDER line
Confirm that RECURSE and EXPORT boxes are checked. Then select IMAGE FORMAT from pull-down menu. Be sure to select format “w/scale marker.” PC TIFF is recommended (“16 Bits” is not recommended as not all programs can read this image format). If notations have been made on the image (text box, arrows, etc.) use “w/overlays” to have these included in the .tif image. If exporting SPECTRA or PROFILES, check the appropriate boxes instead and select the desired export format.
Close OPTIONS window by clicking OK. Then begin batch conversion by clicking EXPORT from the FOLDER EXPORT tool. Each image will be opened in turn during conversion.
6. ANALYSIS Mode
ANALYSIS mode has many functions, some of which are briefly described in a general way below. The HELP menu provides full information about these functions.
The menu commands across the top of the ANALYSIS window permit manipulating multiple aspects of image/data panes, including specific operations for EDX analysis.
On the left side of the ANALYSIS window are “shortcut” tabs that enable image processing, data extraction, EDX analysis, and other functions. Clicking on the tab name will open the commands for that function.
On the right side of the ANALYSIS window are display enhancement and information tools.
Across the bottom of the ANALYSIS window are tools for moving and marking panes with “overlays” (e.g. lines, boxes and text.)
NOTE: While it is possible to acquire images and spectra in ANALYSIS mode, it is not recommended to do so. ANALYSIS mode is not optimized for acquisition and the TIA program will frequently “freeze” or “crash” when attempting to acquire data in ANALYSIS mode.
Using Tecnai CCD Camera
Important Information: The CCD camera is sensitive to the electron beam and can be PERMANENTLY DAMAGED if the electron beam remains on the camera for extended time. Always: Retract Camera when done Searching or Acquiring. DO NOT Condense Beam to Cross-Over while CCD Camera is Inserted. Use BEAM STOPPER when viewing Diffraction Patterns. CLOSE Column Valves before leaving room.
Image and Spectrum acquisition is controlled by the Tecnai UI software on the left-hand monitor, but they are displayed in the TIA program on the right-hand monitor. See Using TIA for more info.
1. Find ROI. Locate the region of interest on the sample by viewing the screen through the TEM window.
2. Insert Camera. From CAMERA tab on the Tecnai UI insert CCD camera by clicking INSERT button. (Button Yellow = In; Button Grey = Out.)
INTEGRATION TIME (i.e. exposure time) can be selected independently for SEARCH, PREVIEW, or ACQUIRE while the respective button is active.
To prevent a bright band appearing on the left side of the image, as in example image shown at right, cover the TEM viewing window.
3. Compose Image. Click SEARCH button to begin viewing image on TIA monitor. Select desired MAGNIFICATION, FOCUS, and center the ROI with joystick.
4. Fine FOCUS. Use PREVIEW button to open a magnified view of image for focusing.
5. Capture Image. Select ACQUIRE to capture an image to save or analyze.
6. Retract CCD/Camera. When done with ACQUIRE, retract the camera from the beam path by clicking INSERT. (Button Yellow = In; Button Grey = Out.)
7. Save Image. Save desired images to appropriate folder on drive C. Convert images to .tif using FOLDER EXPORT. See USING TIA for more details. Gain Reference. (Optional) If image has much background noise that is not part of the sample, click arrow on upper right of CCD/Camera workset to open SETTINGS window. Click BIAS/GAIN tab.
Before beginning Gain Reference it is essential that nothing but vacuum be imaged by the camera during gain reference, not just nothing interesting! Any features in the image will be saved as part of the reference image and subtracted from all subsequent images.
Move stage to a hole in the sample so nothing is in the camera view. If not possible, move sample holder to Parking Position.
PARKING THE HOLDER. Close column vacuum valves FIRST by clicking COL. VAVLES CLOSED. Pull the sample holder straight out from the microscope ~4 cm, then place the Parking Position Wooden Block into position and allow the holder to rest against the wood block. Open COL. VALVES now. The block holds the sample rod out of the path of the electron beam so the camera records an image of the empty vacuum. Remember to close COL. VALVES before moving sample holder rod in or out. Remove the Parking Block by pulling the holder out slightly, remove Block, then slowly insert the sample holder fully into the microscope.
Confirm that there is nothing in view in the camera image and the illumination is uniformly bright on the camera while in SEARCH. Select ALL GAIN from the Gain Reference Acquisition section and wait for the gain reference images to be collected.
Basic EDX
1. Begin by opening the Windows START menu, then select GenesisRTEM. The RTEM Control window will open.
2. Be sure that the Objective Aperture is removed from beam path before continuing.
3. Find an area of interest, A-tilt the holder between 5-20° positive (single-tilt holder to 15-20°.) Condense the beam as required to illuminate the region of interest only. Remember, whatever the electron beam illuminates will produce X-rays.
4. Select the EDX workset window. Set LIVE TIME (normally >100 sec.)
5. Insert the EDX detector by clicking the IN button on RTEM. If the X-ray count-rate is too high, the detector will automatically move to the OUT position and the OUT button will display HI- CPS. Use smaller spot size to reduce the count-rate.
6. Click ACQUIRE on EDX workset. A live spectrum will be displayed in the TIA window. If COUNTS and/or DEAD TIME are high (indicated by red dead time background), reduce count-rate by changing to a higher SPOT SIZE number (= smaller spot size.) Ideal DEAD TIME is <30%.
7. Adjust the scale on the EDX spectrum by dragging the mouse on the spectrum scale.
8. When done collecting spectrum, remove detector by clicking OUT on RTEM. Do this especially before moving the sample to a new location.
TIA Acquire Window:
9. Tools for labeling spectrum peaks, performing spectrum quantification, etc. are located on the right of the TIA Acquire window. Move the cursor over the icons to learn their name and function. The most commonly used EDX tools are:
EDX Quant Setup EDX Peak ID EDX Background Correction EDX Peak Fit EDX Quantify EDX Clear Quant Setup EDX Auto Map EDX Generate Maps/Profiles Quantifying EDX Spectra
Before attempting to quantify any EDX spectra, the User should be familiar with the methods used in EDX quantification and their limitations and artifacts. Accepting the output of the EDAX software without critical examination will often lead to misleading results (at best) and erroneous results at worst. The following assumes the User is knowledgeable about K-factors, Absorption, Fluorescence, false-peaks, precision, accuracy, etc.
1. Switch TIA to ANALYSIS mode (see USING TIA for more information.)
2. Select EDX QUANT from the shortcut tabs on left side of window. Click PEAK ID for software to automatically label each peak. Check the IDs for accuracy. The software will often mislabel peaks or ID peaks that do not actually exist in the specimen.
3. Open PERIODIC TABLE from VIEW menu at top of window. Table will open in pane below the spectrum.
4. Confirm the MARKERS tab at bottom of table is selected. Right-click on any element and select/deselect SHOW to include/remove the ID label from the spectrum.
5. Select the ROI tab at bottom of table, and right-click any element to include/remove from the list of elements to be quantified.
6. Click QUANTIFY for software to automatically calculate elemental concentrations from EDX peaks. Note the exaggerated precision (4 or 5 significant digits!) Use QUANT SETUP to change setup parameters for quantification. Click CORRECT BACKGROUND and FIT PEAKS before QUANTIFY to recalculate quantification table.
STEM: Scanning TEM
INTRODUCTION: In STEM mode images are acquired by moving a focused beam in a raster across the specimen and collecting a signal at each pixel. As the beam is scanned across the specimen in a rectangular raster, the image is built up over time as the signal changes from pixel to pixel. The signals generated by the interaction between the electron beam and the specimen will vary according to specimen characteristics such as material type (composition and structure), orientation (diffraction from crystals), and topography.
Various detectors are positioned below the specimen to receive the signals. Undiffracted beams strike the Bright Field (BF) detector, while diffracted beams strike the Annular Dark Field (ADF) detector. On some STEM microscopes a High Angle ADF (HAADF) collects the highly scattered Z-contrast beams. NOTE: The Tecnai T12 is not equipped with a HAADF.
Note that as the beam is rastered across the specimen the deflection coils first bend the incident beam from the optic axis in such a way that it intersects the specimen in a normal direction. Then the beam is deflected back to the optic axis so as to strike the stationary detectors at normal incidence (see green beam path in above image). Because of the complex deflection angles, it is necessary that the microscope column alignments be performed carefully to obtain the best STEM image results.
1. Insert and center Condenser Aperture number 3. Remove Objective Aperture.
2. From STEM workset, set microscope to STEM mode by clicking STEM button (turns yellow.)
3. Click SEARCH. Click Diffraction Center in DIRECT ALIGNMENTS. Center beam on viewing screen with MF X & MF Y.
4. Lift the TEM screen by pressing SCREEN LIFT button located around base of TEM column on left or by identifying one of the L/R panel buttons as ‘Screen Lift.’ 5. Select DETECTOR CENTER in DIRECT ALIGNMENTS. Align by maximizing intensity of STEM image using MF X & MF Y.
6. Focus image. A reduced image window opens when FOCUS button is clicked.
7. Press STIGMATOR button and carefully stigmate the Condenser Lens. (Make the image sharp.) 8. Adjust contrast and brightness (click AUTO C/B) as necessary to obtain best image. To change scan rate (Frame Time), tab-out by clicking arrow in upper right box and make changes in new window.
9. Move the ROTATION slider to change the orientation of the displayed image. Click BLANK to prevent the beam from reaching the specimen. (When not searching the beam remains stationary at the center of the screen. This focused stationary beam may damage the specimen.)
10. To view Dark Field image, tab-out on the STEM DETECTOR window and make one of the detector choices DF. It is possible to view BF and DF images simultaneously while in STEM mode. Simply designate one detector BF and a second DF.
11. Changing CAMERA LENGTH can result in dramatic changes in STEM images, particularly DF images. Different diffracted intensities will strike the ADF detector at different Camera Length settings. At low Camera Length settings it is possible to simulate the effect of a HAADF detector. At high Camera Length settings the undiffracted disc will strike the ADF and cause the DF image to look like a BF image. Re-do Direct Alignments, especially Detector Alignment when changing Camera Length. At small Camera Length settings, the beam may not be able to reach the STEM detector.
Manual CONTRAST and BRIGHTNESS controls, as well as AUTO C/B, are located on the STEM DETECTOR window.
STEM images do not have resolution as high as TEM images. It may be possible to improve the STEM image resolution by operating at a higher spot size value (smaller spot size.) Confirm that Direct Alignments have not changed after changing spot size.
EDX with STEM: Spectrum Images and Profiles
1. Set microscope to STEM mode.
2. Lift the TEM screen and obtain a STEM image in the TIA window by clicking SEARCH. Focus, adjust C/B, Align, etc, as necessary to obtain best image.
3. While searching, open EDX window and begin acquiring a spectrum.
4. ID peaks. Open the Periodic Table and change peak Markers and ROIs if needed.
5. ACQUIRE STEM image.
6. On EDX page, from the EXPERIMENTS workset, select SPECTRUMCOLLECTION from the pull-down menu. 7. From the SELECT EXPERIMENT pull-down menu select the type of data to collect. SPECTRUM POSITION collects EDX spectrum from individual points; SPECTRUM PROFILE collects spectra along a designated line; SPECTRUM IMAGE collects spectra from an area and produces “elemental maps.” 8. Define the PROFILE SIZE (i.e. number of pixels) and DWELL TIME at each pixel. Then select YES for ACQUIRE EDX SPECTRA. If collecting a spectrum image also select YES for ACQUIRE STEM IMAGE. 9. In the TIA window the points, profile line or image box will be drawn on the previously acquired STEM image. These can be positioned and resized independently of the parameters defined in the EXPERIMENTS workset. 10. Confirm that the objective aperture in not inserted. Open Genesis RTEM and insert EDX detector. Press ACQUIRE button on EXPERIMENTS window when ready. 11. Note the displayed acquire time. Stop Acquire and adjust Settings if the time is too long. Remember that image drift is possible during long scans, and can be corrected by choosing from the experiment pull-down list. Remember also that shorter dwell times means fewer X- ray counts, which means poorer results. 12. When the Acquire is finished, save the resulting spectra file. 13. Select AUTOMAP from the left-hand column of TIA in ANALYSIS MODE. Open SETTINGS and define the parameters, then click GENERATE. Examples appear on next page.
Spectrum Profile (Line)
Spectrum Image (Maps)
End-of-Session Steps
1. Set Magnification to 23 000 x. Spread beam to fill viewing screen.
2. Remove Objective and Selected Area Apertures by moving lever to right.
3. Open SETUP workset. Close valves by clicking COL VALVES CLOSED. (Grey = Open; Yellow = Closed)
4. Click FILAMENT button to turn off (Yellow = On; Grey = Off.) Wait for FILAMENT cooling to complete (Blue line gone.)
5. Click HIGH TENSION to turn off (Yellow = On; Grey = Off.)
6. Open STAGE workset; tab-out STAGE2. Click “HOLDER” in ‘Reset’ area to set stage positions to zero.
7. Remove sample holder:
- Pull holder straight out until stop.
- Turn holder clockwise toward CLOSED until stop (do NOT pull while turning.)
- Pull holder fully straight out (do not hesitate or move holder inward while pulling out.)
- If using double-tilt holder, disconnect cable.
8. Close all programs.
9. Log out of Windows.
10. Remove sample and return sample holder to storage box.
11. Fill out log book on desk and log sheet on wall.
12. Night User ONLY:
- Remove LN2 dewar.
- Start CRYO CYCLE. START AFTER = 10 DURATION = 180