Standard Operating Procedure (SOP) - Thermo Fisher Scientific - Phenom XL G2 Desktop Scanning Electron Microscope (SEM) - Basic Operations

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Figure 1. Overview of SEM System

Section 1: Introduction to Equipment

The Phenom XL G2 Scanning Electron Microscope (SEM) provides users with high resolution electron
microscopy images in just a few minutes. Users can load many sample stubs into the sample holder and
navigate around each sample in the Phenom User Interface application. Features include Automated Image Mapping (AIM), returning to saved positions on samples, Energy-dispersive X-ray Spectroscopy (EDS) and Live EDS allowing for quick elemental identification and EDS mapping. There are two main modes, NavCam (light optical) and SEM (electron optical). Within SEM mode, the EDS, Live EDS and AIM features can be utilized. Some of the other key features are:

• Accommodates sample sizes up to 100 mm x 100 mm with 35 mm height

• Sample stubs with 0.5” diameter

• Electron optical magnification range of 160 - 200,000x

• Light optical magnification range of 3 - 16x

• Provides a resolution of <10 nm

• Acceleration voltages of 5 kV, 10 kV and 15 kV

• Able to analyze particle samples, polymers, metals, biological samples and more

Section 2: Using the Phenom XL G2 Desktop SEM

2.1: Turning on and logging into the system

1. Login to the desktop PC. The Phenom User Interface application should already be running. If it is not, launch the Phenom User Interface application on the desktop as seen in Figure 2.

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   Figure 2. Application Icon

2. The instrument will likely be in standby mode. If this is the case, click on the dialogue box on the bottom left corner of the screen to wake up the instrument as seen in Figure 3.

2.2: Sample preparation

1. Grab a sample stub that best suits the sample analyses requirements.

2. Apply a piece of double sided carbon tape to the sample stub. Place the sample onto the exposed adhesive. Ensure the sample is firmly attached to the stub, as seen in Figure 4.

3. Unload the sample tray from the instrument by clicking the load/unload button on the top left of the screen as seen in Figure 5.

4. Take out the sample tray and turn the height adjustment wheel at the front counter-clockwise, as seen in Figure 6, until the mounting surface is in the highest position as seen in Figure 7.

5. Insert the stub pin into one of the holes on the mounting surface, using the sample loading tweezers. Ensure the stubs flat lower surface is seated on the mounting surface of the sample tray.

6. Lower the sample by turning the height adjustment wheel clockwise until the top of the sample is flush with the top of the sample tray as seen in Figure 8 and 9. Afterwards, turn the height adjustment wheel clockwise six more notches, lowering the sample platform.
   
   

    

    

7. Gently insert the sample tray into the loading bay. Ensure that the end of the sample tray going into the loading bay is slopped slightly downward as seen in Figure 10. Do not force the sample tray when loading, there should be no resistance when sliding it in. See Figure 11 for how the sample tray looks when being loaded and Figure 12 for how the sample tray looks when it is fully loaded into the loading bay.

8. Once loaded, close the loading bay door by clicking the load/unload button on the top left of the screen as seen in Figure 5.

2.3: Navigating the user interface

After loading the sample the NavCam Live view will be displayed. This view also contains various buttons along the perimeter of the screen as seen in Figure 13.

1. Imaging controls for the NavCam (light optical) and SEM (electron optical) mode can be adjusted with the side panel. To open the side panel, left click the arrow button on the center right of the screen and drag the sliders to adjust values as seen in Figure 14. Above the sliders, a zoomed out screenshot of the entire sample tray is displayed along with a blue crosshair which signifies where the Live view is currently centered around. Left clicking anywhere on the sample tray screenshot will update the location that the Live view is centered around. There is also the capability to create and access saved positions, located below the sliders. The same optical adjustments can also be performed by using the three buttons on the bottom left of the screen, as seen in Figure 13.
   
   

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   1. When in NavCam mode the buttons behave as outlined below

      1. Left clicking the magnification icon and scrolling with the mouse will adjust values.

      2. Left clicking the contrast/brightness icon and scrolling with the mouse will adjust one of the two values. Once selected, an additional left click toggles between contrast and brightness selection.

      3. Left clicking the focus icon and scrolling with the mouse will adjust values. Once selected, an additional left click toggles between fine and coarse adjustment.
         
         

   2. When in SEM mode the buttons behave as outlined below

      1. Left clicking the magnification icon and scrolling with the mouse will adjust values.

      2. Right clicking the brightness/contract icon brings up the option to switch between ‘Auto’ and ‘Manual’ mode, as seen in Figure 15. When ‘Auto’ is selected, left clicking the icon automatically adjusts the brightness and contrast values. When in ‘Manual’ mode, left clicking the icon toggles between contrast and brightness selection, and scrolling with the mouse adjusts values.
         
         

          

          

      3. Right clicking the focus icon brings up the option to switch between ‘Auto’, ‘Manual’ and ‘Focus in selected area’ mode, as seen in Figure 16. When in ‘focus in selected area' mode a blue rectangle overlays the sample. All of the changes to the optical parameters will only be applied to the area inside of the rectangle. The size and shape of the rectangle can be adjusted by left clicking any four corners and dragging the mouse. To exit this mode, left click anywhere outside of the blue rectangle. When in ‘Auto’ mode left clicking the focus icon automatically adjusts the focus values. When in ‘Manual’ mode left clicking the focus icon toggles between fine and coarse adjustments and scrolling with the mouse adjusts values.

2. To adjust the system, Live image and acquisition settings for SEM mode, open the top menu by clicking the arrow button at the center top of the screen as seen in Figure 13. This will open up the menu as seen in Figure 17.
   
   

    

   1. Accelerating voltage

      1. 5kV: Most surface sensitive, useful for thin samples

      2. 10kV: Best resolution

      3. 15kV: Highest interaction volume but produces the most noise

   2. Beam intensity

      1. Low: Best resolution at high magnification (20kx)

         1. Use with higher vacuum & higher accelerating voltage

      2. Image: Best for 1kx to 8kx

      3. Point: Best for Live EDS and point EDS

      4. Map: Best resolution at very low magnification (1kx) and EDS mapping, use with low vacuum & high working distance

   3. Detectors

      1. BSD (Back Scatter Detector) Full

         1. Z-contrast, heavier atoms appear brighter

      2. Topo A and Topo B

         1. Both use a different half of BSD to give topographical features

   4. Chamber Vacuum Pressure

      1. High (1 Pa) - best for conductive samples, this includes gold-coated samples

      2. Medium (10 Pa) - best for semiconductors

      3. Low (60 Pa) - best for non-conductive samples

   5. Averaging (for both “Live” and “Acquisition”)

      1. Live: No averaging; most noise visible, but fastest refresh rate/image acquisition

      2. Medium: Averages 4 frames; good for Live image and Acquired image

      3. High: Averages 16 frames; maximum necessary for both the Live and Acquired
         image

      4. Best: Averages 32 frames; suitable when encountering extreme amounts of noise

   6. Scan Size (under both “Live” and “Acquisition”)

      1. Different available options between Live and Acquisition

      2. Higher scan size increases quality, but reduces refresh rate in the Live image and increases acquisition time

3. To view all of the selected imaging parameters and settings previously discussed, open the bottom menu by left clicking the arrow button on the center bottom of the screen as seen in Figure 13. To change the name of the image being captured, click the blue text on the right labelled ‘Sample’, as seen in Figure 18, and enter a unique name.

4. After loading the sample tray the Live view is selected by default and is in NavCam mode. To select the Live view, left click Live/Overview Icon in the top right corner of the screen as seen in Figure 21. Live view is where images can be captured on both NavCam and SEM mode. The functionality is the exact same for the live view when in SEM and NavCam mode.
   
   To make minor adjustments to the location of the camera left click anywhere on the sample. The view will shift so it centers around the area that was clicked. To make bigger movements to the camera, left click the area of interest on the light optical screenshot of the sample tray located at the top of the side panel menu as described in section 2.3.1. Once optical parameters have been adjusted, click on the camera icon located at the center left of the screen, as seen in Figure 19, to capture an image.
   
   

   1. To enter SEM mode click on the move to electron beam icon at the top left of the screen as seen in Figure 20. To get out of SEM mode, click the unload/load icon as seen in Figure 5.

6. To use the Automated Image Mapping (AIM) feature, the user must already be in SEM mode. Left click the icon on the top right of the screen as seen in Figure 22. This feature allows for capturing a large area of the sample without sacrificing the resolution, by taking multiple images that when stitched together covers the defined area of interest. Once selected, the option to use a rectangle or polygon shape will show up on the center left of the screen, as seen in Figure 23, and the AIM settings will display on the right side of the screen as seen in Figure 24. Left click the desired shape to select it. For the rectangle, left click, hold and drag to highlight the area of interest. For the polygon, left click as many times necessary to create the desired shape and right click for the last point of the shape, this will complete the shape. The mouse scroll always controls the magnification in this mode. Afterwards, select the desired settings for the AIM function.

   1. In random, the following options are provided:

      1. Number of images to be captured (number of tiles)

      2. How magnified the images should be (tile field of view)

      3. Which detector to be used

   2. In stitched, select how magnified each tile should be (tile field of view)

      1. Shows how many images will be patched together based on the tile field of view selected

   3. Once all relevant parameters are entered, click start and enter a project title

5. To use the EDS function, the user must already be in SEM mode. Click on the icon seen at Figure 25.

   1. For best results, use a 15kV accelerating voltage

   2. Analysis time and settings can be customized by left clicking the cog wheel/setting icon on the top left of the screen, as seen in Figure 13, and selecting the EID tab on the left side of the menu as seen in Figure 27.

   3. On the left-side toolbar, several new icons appear for EDS, as seen in Figure 26:

      1. Point (yellow) - Left click on the image after selecting the icon

      2. Region (red) - Left click and drag after selecting the icon

      3. Line (blue) - Left click and drag after selecting the icon

      4. Map (green) - Left click on the image after selecting the icon to analyze the entire image, or left click and drag to analyze a particular area of the image

         1. For the Map option, several elemental maps will appear below a captured SEM image. Ctrl + Click provides overlays of these maps to determine elemental distribution across the mapped region, as seen in Figure 32 and 33.

   4. Once one of these tools are selected, they can be applied to the area of interest within the image view on the top left of the screen as seen in Figure 31.

   5. After collecting a sufficient amount of spectra, click the square ‘Stop’ icon (pink) as seen in Figure 26. Hover over the final spectra and scroll to zoom along the x-axis. Hover the cursor over peaks to see
      their energy and counts in the bottom right of the spectra as seen in Figure 28.

   6. Right-clicking and selecting “Lock highlight” will show possible emission lines in a box to the right of the spectra as seen in Figure 29. To unlock, right click again and select “Unlock highlight”.

   7. Left-clicking on elements in the periodic table shows their emission lines on the spectrum. Right-clicking allows for the option to include or exclude specific elements from the composition calculation as seen in Figure 30.

6. To use Live EDS left click the 'LIVE EDS' icon, as seen in Figure 36, located on the top right of the screen. This is a fast method typically used to verify the composition at certain points. Click on the crosshair icon at the center left of the screen so it turns blue, as seen in Figure 37, and left click a point of interest on the live sample view to perform EDS at that spot. Once enough data has been captured click the square icon, as seen in Figure 37, to stop the live EDS function. Data is displayed and manipulated in the same manner as the EDS function, as described in 2.3.5. Navigating to an area of interest works the same way as the Live view, as described in 2.3.4.

7. To view images, their associated metadata and the directory they were saved in click on the gallery icon at the top right of the screen, as seen in Figure 38. The latest image captures will be displayed as seen in Figure 39. Click on the displayed image on the top left of the screen as seen in Figure 39. This will blow up the image, as seen in Figure 40, and provide the ability to add measurements and annotations to the image. Click either of the highlighted symbols in the top left of the screen, as seen in Figure 40, and left click the areas of the image that these features should be applied to. To change the save location and name of acquired images, click on the cog wheel/settings icon on the top left of the screen, as seen in Figure 39, select the ‘Customize’ tab on the left of the menu, then the Acquisition tab at the top of the menu, and edit the ‘Label’ and ‘Location’ values as required, as seen in Figure 41.

2.4: Unloading and Shutting Down

1. Once finished using the instrument, unload the sample tray, as described in Section 2.2, remove the samples, and load the sample tray back into the loading bay as described in Section 2.2.

2. Go to Settings > Status and set the instrument to Standby in the top left corner of the screen as seen in Figure 42.