red = needs attention

italics = drafty language

blue = expanded information for the User Guide (remove for the quick-start guide)

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Figure 1. The SNE-4500M Plus with numbers denoting the key interactions for a user. Image from the SNE-4500M Plus user manual at www.seceng.co.kr

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Our SEC SEM is equiped with a Bruker Quantax electron dispersive spectrometer (EDS) attachment, which allows for qualitative to semi-quantitative chemical microanalysis of geological materials. To use the SEC SEM with Bruker EDS attachment, please first follow the instructions in this guide, and then proceed to the SEC SEM with Bruker EDS Quick Start Guide.

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What are For additional resources (link them here)Image Removed, please refer to the SEC SNE-4500M Plus Quickstart Guide (from the manufacturer) or the manufacturer's manual.

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II. Procedure

A. Sample preparation and loading the sample

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1. Push the ‘power switch’ on the left-hand side of the microscope (Figure 1.2).  
2. Launch the Nanoeye software icon (Figure 2) on the desktop to launch the program. Occasionally the software will fail to launch, due to issues with communication. Restarting the computer will reset the COM ports and should allow the user to launch the software.
   1. Note that the Nanoeye software will launch under the Admin user account, so anything saved to the Desktop will not be accessible to anyone logged into the typical user account. For that reason, save all data into the C:\\SEM directory.
3. Prepare your sample for SEM analysis. Depending on the size and material, this may require gold/palladium sputter coating or carbon-coating (used for EDS). For sputter/carbon coater instructions, see the Sputter Coating Quick Start Guide.
   
   1. Common sample types include: stubs, with or without sputter coating; thin-sections with carbon coating; grain-mounts with carbon coating.
   2. Somewhere need to explain that coating is to prevent charging, which is especially common with thin-sections. Show a picture. Can copy some of the text out of the Hitachi SEM user guide
4. Using the jig (Figure 3), measure the size of the specimen including mount. Use the gradations of the horizontal grid on the jig to measure the diameter of the specimen and the vertical grid for the specimen height. If you are using older 3.2mm style stubs with a narrow attachment post, there is an adapter available to fit into the stage. Make sure to measure dimensions with the adapter attached.
5. Important: use compressed air to blow off any loose material on the sample that otherwise could be mobilized within the vacuum and damage the detectors.

Image RemovedFigure 2. Nanoeye software icon

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Figure 3. The mounting jig used to measure the width and height of the sample.

Loading the sample into the SEM

1. When not in use, the SEM is typically left under vacuum. Thus, to load a sample, the SEM needs to be pumped with air. Press the Exchange button on the front of the SEM, which will activate the vacuum and evacuate the chamber. Press the Exchange button again to initiate the SEM filling with air. The button LED light indicates the inner vacuum status of the chamber, and the signals are: 
   1. Light Off: Vacuum is not applied
   2. Light On: Vacuum is applied
   3. Slow blinking: Vacuum is being released
   4. Fast blinking:  Vacuum is being applied
2. Follow the LED strip on the front of the SEM which is a progress bar, with a fully illuminating LED strip indicating that the SEM is at vacuum. Listen for a double? beep, which indicates that the vacuum has fully been released.
3. Fully open the stage door and click the ‘Calibration’ button to initiate the stage motor calibration (Figure 4). This should take around 3 minutes, and all motor controls will go back to the home position when done and no numbers should be yellow.? Occasionally the motor will get stuck at its limit switch and the value will stay yellow, in which case you can run the Calibration routine again.
4. In the Nanoeye software, click the “Sample Information” text box (Figure 4, red rectangle), enter the height and width values, then press Enter. After the height is entered, the Z-axis will automatically lower the stage to accommodate for the entered height to a distance of Z = [Entered height]. Important: The command will not be registered if the user fails to hit Enter, and the user runs the risk of colliding the sample into one of the detectors.

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Figure 4. Nanoeye window showing the sample preparation commands. Enter the height and width into the Sample Information (red box).

5. Insert the stub to the stage:

1. 1. Use a 1.5 mm allen wrench to carefully loosen the set screw, then insert the mount with the specimen to the stage, and tighten the set screw (Figure 5).
   2. Important: Ensure that stub (+/- adapter) is seated fully into the stage mount. Use caution when loading samples to prevent accidental collision with detectors. Gloves should be worn when handling any components/sample material that goes into the vacuum chamber.
   3. Skin oil and loose debris can damage the detector. Stubs should be prepared with gloves. For thin-sections and grain mounts, they should be wiped with isopropanol. It may be beneficial to leave a grain mount overnight in a vacuum or dessicator cabinet, as the larger quantity of epoxy can de-gas water vapor and other volatiles.
   4. If you lose the set-screw for the stage, there are a few spares in a small ziploc bag in the clear case with SEC SEM accessories.

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Figure 5. Mounting the stub to the stage.

6. Capture the specimen image to aid in navigation of the stub. To do so:

1. 1. 1. 1. Slide the door halfway closed to the point where the door catches and clicks.
         2. In Nanoeye, click the “Camera” button and the screen display will show the specimen (Figure 6). 
         3. With the camera activated, right click the “Camera” button to activate the brightness/contrast menu (Figure 6).
            Click the camera button again to take an image. 

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Figure 6. Display windows of the navigational camera operations.

 7. Close the chamber door gently and push the Exchange button (Figure 1.3) to put the chamber under vacuum. Gently press the door into the SEM to aid the seal as the vacuum begins to pump down.

B. Turning on the beam

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Once the machine is under vacuum (indicated by the SEM beeping twice), navigate to the Operation Panel (Figure 7). IMAGE GOES HERE 

Select an accelerating voltage (range: 1 to 30 kV; see table 1 for working guide).

Table 1. Working guide for accelerating voltage 

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Figure 7. Operational start-up window

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1. the secondary electron (SE) detector, which returns an image of the topography of the sample's surface and is generally used for micropaleontological identification with SEM stubs
2. or the back-scattered electron (BSE) detector, which returns a pixel value based on the average atomic number (Z) of a given material and is generally used on thin sections or grain mounts.
3. User guide can have a more in-depth explanation of the two different detectors and show samples of SE and BSE images, how they work.

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C. Image refinement

Basic software controls

1. Once the beam is on, you can navigate around the sample in the x-y direction by double-clicking on the SEM display or sample map/camera screen. Can also change the x-y.
2. Cautionary note about changing the z, rotation
3. Magnification can be adjusted using the mouse wheel.

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Figure 8. The left-hand panel consists of the focusing, beam area, signal processing (Image area) and scan rates(Scan area) controls

Focusing the microscope

1. Click the buttons under “Focus” area to adjust the focus (Figure 10) until an image is visible. This should be at approximately _______________. <<<need to describe how to use the UI
2. In several steps, increase the magnification then adjust the Focus first using the Coarse Focus Adjustment arrows, then the Fine Focus Adjustment arrows. Do this until you have reached a good focus at the desired magnification.

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Figure 10. the focus adjustment window, with descriptions of each button

Tips for focusing the microscope

1. Tips.....
2. If the screen wobbles significantly in either horizontal or vertical direction during focus adjustment, this indicates that the variable aperture strip may be out of alignment. Let a technician know and they will manually adjust the aperture’s alignment using knobs on the side of the instrument. Include instructions

Improving the image

Tighten the beam area (spot size)

1. If the image looks dark, adjust the “Spot Size” (the lower the % value, the greater the amount of beam and the brighter the image). 
2. Reduce the spot size of the beam when the amount of focused beam becomes larger. This option is suitable for ultra-fine images (the higher the % value, the tighter the beam focus, but the image will also become darker).

Adjust the brightness/contrast

Change the scan speed

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Figure 9. Various scanning speeds. Button in the top right is the measuring tool, where you can measure the distance and angle between two points, measure a marked area and annotate

Get a technician

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Advanced image correction (for technicians)

Correct the beam shape

Align the strip aperture

Adjust the stigmation

Align the beam

Recommended settings

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• 1-5 kV (higher if sample is Au/Pd coated)

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• 5-10 kV

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• 10-20 kV

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• 10-20 kV

Additional tips

• Dealing with movement
  
  • Sea state
  • Jostling the table
• For high magnification (>5000x; unlikely at sea conditions)
  • Change the variable strip aperture
  • Increase the Spot Size (higher number)
  • Change the accelerating voltage up or down
  • Adjust the stigmation
  • Focus the microscope/decrease the scan speed

If the sample is charging

An explanation of what charging means and why it's bad

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1. 1. The purpose of carbon and metal sputter coating is to ensure that the entire surface of the sample is conductive, to avoid the accumulation of electrons on the surface of the sample. When this occurs, the electrons on the sample repel the electrons of the beam, creating localized image distortion. "Charging" tends to be progressive, so if it is encountered, the user should take steps to resolve it. These steps could include:
      1. Decreasing the magnification
      2. Changing the spot size in order to make the beam more diffuse
      3. Changing (typically decreasing) the accelerating voltage
      4. Taking the sample out in order to apply a thicker carbon/sputter coat
      5. (For SEM imaging only) changing the microscope set-up to low vacuum.
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2. Using the jig (Figure 3), measure the size of the specimen including mount. Use the gradations of the horizontal grid on the jig to measure the diameter of the specimen and the vertical grid for the specimen height. If you are using older 3.2mm style stubs with a narrow attachment post, there is an adapter available to fit into the stage. Make sure to measure dimensions with the adapter attached.
3. Important: use compressed air to blow off any loose material on the sample that otherwise could be mobilized within the vacuum and damage the detectors.

Image AddedFigure 2. Nanoeye software icon

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Figure 3. The mounting jig used to measure the width and height of the sample.

Loading the sample into the SEM

1. When not in use, the SEM is typically left under vacuum. Thus, to load a sample, the SEM needs to be pumped with air. Press the Exchange button on the front of the SEM, which will activate the vacuum and evacuate the chamber. Press the Exchange button again to initiate the SEM filling with air. The button LED light indicates the inner vacuum status of the chamber, and the signals are: 
   1. Light Off: Vacuum is not applied
   2. Light On: Vacuum is applied
   3. Slow blinking: Vacuum is being released
   4. Fast blinking:  Vacuum is being applied
2. Follow the LED strip on the front of the SEM which is a progress bar, with a fully illuminating LED strip indicating that the SEM is at vacuum. Listen for a double? beep, which indicates that the vacuum has fully been released.
3. Fully open the stage door and click the ‘Calibration’ button to initiate the stage motor calibration (Figure 4). This should take around 3 minutes, and all motor controls will go back to the home position when done and no numbers should be yellow.? Occasionally the motor will get stuck at its limit switch and the value will stay yellow, in which case you can run the Calibration routine again.
4. In the Nanoeye software, click the “Sample Information” text box (Figure 4, red rectangle), enter the height and width values, then press Enter. After the height is entered, the Z-axis will automatically lower the stage to accommodate for the entered height to a distance of Z = [Entered height]. Important: The command will not be registered if the user fails to hit Enter, and the user runs the risk of colliding the sample into one of the detectors.

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Figure 4. Nanoeye window showing the sample preparation commands. Enter the height and width into the Sample Information (red box).

5. Insert the stub to the stage:

1. 1. Use a 1.5 mm allen wrench to carefully loosen the set screw, then insert the mount with the specimen to the stage, and tighten the set screw (Figure 5).
   2. Important: Ensure that stub (+/- adapter) is seated fully into the stage mount. Use caution when loading samples to prevent accidental collision with detectors. Gloves should be worn when handling any components/sample material that goes into the vacuum chamber.
   3. Skin oil and loose debris can damage the detector. Stubs should be prepared with gloves. For thin-sections and grain mounts, they should be wiped with isopropanol. It may be beneficial to leave a grain mount overnight in a vacuum or dessicator cabinet, as the larger quantity of epoxy can de-gas water vapor and other volatiles.
   4. If you lose the set-screw for the stage, there are a few spares in a small ziploc bag in the clear case with SEC SEM accessories.

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Figure 5. Mounting the stub to the stage.

6. Capture the specimen image to aid in navigation of the stub. To do so:

1. 1. 1. 1. Slide the door halfway closed to the point where the door catches and clicks.
         2. In Nanoeye, click the “Camera” button and the screen display will show the specimen (Figure 6). 
         3. With the camera activated, right click the “Camera” button to activate the brightness/contrast menu (Figure 6).
            Click the camera button again to take an image. 

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Figure 6. Display windows of the navigational camera operations.

 7. Close the chamber door gently and push the Exchange button (Figure 1.3) to put the chamber under vacuum. Gently press the door into the SEM to aid the seal as the vacuum begins to pump down.

B. Turning on the beam

1. Once the machine is under vacuum (indicated by the SEM beeping twice), navigate to the Operation Panel (Figure 7).

2. Select an accelerating voltage (range: 1 to 30 kV; see table 1 for working guide).

   1-5 kV	Delicate or uncoated samples (e.g. microfossils)
   5-10 kV	Coated biological samples (e.g. Au coated microfossils, recommended)
   10-30 kV	Carbon-coated thin section samples

   Table 1. Working guide for accelerating voltage 

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   Figure 7. Operational start-up window

   
   

3. Select which detector to use:
   1. the secondary electron (SE) detector, which returns an image of the topography of the sample's surface and is generally used for micropaleontological identification with SEM stubs
   2. or the back-scattered electron (BSE) detector, which returns a pixel value based on the average atomic number (Z) of a given material and is generally used on thin sections or grain mounts.
   3. Image AddedFigure 7.1. The SE detector, the BSE detector and the EDS detector vary in geometry and in the volume/depth of the sample where those electrons originate.
4. Click the START button to generate the electron beam (Figure 7). 
5. Monitor the emission current, which should be around 110uA. If it has diverged by more than ±20 uA, please notify a technician as this either means the filament is about to die or the beam is not stable, and image quality will be subpar.

C. Image refinement

Basic software controls

1. Once the beam is on, you can navigate around the sample in the x-y direction by double-clicking on the SEM display or sample map/camera screen. Can also change the x-y.
2. Cautionary note about changing the z, rotation
3. Magnification can be adjusted using the mouse wheel.

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Figure 8. The right-hand panel consists of the focusing, beam area, signal processing (Image area) and scan rates(Scan area) controls

Focusing the microscope

1. Click the buttons under “Focus” area to adjust the focus (Figure 10) until an image is visible. The focal plane should generally be at approximately WD [mm] = Z - Measured Height.
2. In several steps, increase the magnification then adjust the Focus first using the Coarse Focus Adjustment arrows, then the Fine Focus Adjustment arrows. Do this until you have reached a good focus at the desired magnification.

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Figure 10. the focus adjustment window, with descriptions of each button

Tips for focusing the microscope

1. Avoid using either Autofocus buttons (AW and AF) as they do not generally work.
2. It is useful to first perform coarse focus at a low magnification, before progressively increasing the magnification while performing coarse then fine focus adjustments.
3. Focussing should be performed at either the fastest or second fastest scan speed.
4. Quality of image while using an SEM at sea depends on sea state. Movement of the SEM due to ship motion is generally visible as low period swaying of the sample.
5. Focus slowly. Once the user is performing fine focus adjustments, if it is difficult to find the point of best focus it can be helpful to find the upper and lower bounds where the image starts to degrade on either sides of the "good focus", and then tune the microscope to the midpoint between the upper and lower limits of fine focus.
6. If the focus cannot be found anywhere within a few mm of the approximate focal plane at WD [mm] = Z - Measured Height, it may be helpful to remove the sample from the SEM and re-calibrate the stage to reset the Z axis.
7. If the screen wobbles significantly in either horizontal or vertical direction during focus adjustment in a way that is not due to sea state, this indicates that the variable aperture strip may be out of alignment. Let a technician know and they will manually adjust the aperture’s alignment using knobs on the side of the instrument.
8. If, ultimately, the user follows the above instructions and cannot find a good focus, consult a technician.

Improving the image

Tighten the beam area (spot size)

1. If the image looks dark, adjust the “Spot Size” (the lower the % value, the greater the amount of beam and the brighter the image). In typical usage on the JR, images typically look best around Spot size = 20.

Adjust the brightness/contrast

1. In the Image Area of the SEM software (see figure 8) there are two sliders which allow the user to adjust the brightness and contrast, along with an Auto brightness/contrast button.
2. It is recommended to select the Auto brightness contrast, then change the brightness and contrast sliders to ensure that the image contains a dynamic range of light and dark that show off the features that the user is attempting to image. At lower scan speeds, the user may observe a degree of noise, which may increase the perceived amount of contrast.

Change the scan speed

1. In the Scan Area of the SEM software (see figure 8 and figure 9), the user can change the scan speed to do slower scan speeds.
2. Slower scan speeds have a higher image resolution and take a longer time.
3. Depending on the sea conditions, it may be beneficial to take images at a faster scan speed (which has a higher amount of noise) to minimize distortion due to the ship's motion.

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Figure 9. Various scanning speeds. Button in the top right is the measuring tool, where you can measure the distance and angle between two points, measure a marked area and annotate

Get a technician

Image correction is generally iterative, by going through the above steps. Technicians can perform additional image correction troubleshooting steps. Additionally, oftentimes quality of imaging is dependent on the sea state and it may be beneficial to wait for calmer seas.

Advanced image correction (for technicians)

Align the strip aperture

Technician instructions for adjusting the variable aperture knob are as follows.

1. Focus the microscope and change the brightness/contrast to the point that you can see a prominent feature.
2. Change to moderate magnification (500-2000x)
3. Select the Wobble button. You should begin to see the microscope move in and out of focus. Ideally, the image should go in and out of focus, with very little up-down or left-right motion (or respectively X and Y motion as observed on the screen).
4. Remembering where the knob originally was, slowly adjust one of the knobs on the variable aperture knob. The one on the left corrects left-right/x-axis motion, and the one on the right corrects for up-down/y-axis motion. If the direction you are moving the knob seems to worsen or quicken the motion observed on the screen, go the opposite way.
5. Typically you will first start adjusting one knob, then another. You are ultimately trying to minimize the observed motion that the image experiences.
6. After you are satisfied with the position of the variable aperture strip, de-select Wobble and re-focus the microscope.

The variable aperture knob is used to tighten the electron beam, in order to reach higher levels of magnification. For JR levels of magnification, we only use the largest aperture. On the knob, 4 red lines should be showing. If not, consult a more experienced technician or consult the manufacturer's manual on how to change to the largest aperture.

Note that the knob is quite delicate and should not be adjusted by scientists and all care should be taken to make sure that it isn't jostled.

Adjust the stigmation

Stigmation corrections are generally not necessary at less than 5000x, however if you observe smearing in the image, it is possible that the stigmation controls are incorrect (another possibility is that the sample is charging). To correct the stigmation controls:

1. Going into the Stigmation tab of the Beam Area panel (see figure 8), first note the current values of the Stigmation_x and Stigmation_y sliders. This is helpful to note in case you want to return to the current values after changing the sliders.
2. Choosing first one slider then the other, move the slider up and down with the goal of trying to increase the sharpness.
3. If a sharper image is not reached, change the stigmation sliders back to the values that they were at when you entered the panel, then attempt another image correction.

Align the beam

The beam can be aligned in two ways. Small corrections to the beam position can be done using the software, but for larger corrections, you will need to lift off the top panel of the sem, then adjust the screws that hold the electron gun in place. The instructions are as follows:

1. After having focused the image, go into the Settings tab, first note the current values of the Gun align.
2. Select Auto gun align, which will systematically change the position of the gun, while the software tries to find the brightest possible image. You can also manually adjust the sliders, again looking for the brightest possible image.
3. If the brightest possible image is reached at one of the extremes of either of the sliders (x- or y-alignment), you will have to manually lift off the top panel of the SEM to adjust the screws that hold the electron gun in place. This is done with the SEM on, but note that the electron gun might be hot if you have had the electron beam on for a long period of time. Use caution.
4. The lid of the electron gun is held in place with 4 knobs which should be finger tight. Slightly loosed all knobs, then pick two knobs that are opposite each other.
5. Holding the two knobs that are opposite each other, systematically loosen one knob then tightening the opposite knob in order to move the location of the electron gun. Do this while looking at the image on the screen while trying to find the position with the brightest image.
6. After you have optimized one set of knobs opposite each other, optimize the other set of knobs for the brightest image. This should be iterative to find the best and brightest image.
7. All knobs should be tightened to a light but firm tightness, after adjustment is done.
8. You will likely have to change brightness/contrast of the image after you are done, and you may have to re-align the strip aperture.

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Recommended settings

D. Capture an image

Annotating the image (optional)

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