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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

I. Introduction

The SEC SNE-4500M Plus scanning electron microscope (SEM) captures highly magnified images of a specimen by scanning an electron beam over a sample in a vacuum chamber. An electron gun produces an electron beam, which is concentrated into a fine beam by passing through a series of electromagnetic coils and condenser and objective lens. Electrons are used to bombard the specimen, which produces secondary and back-scattered electrons. These electrons are respectively detected by a secondary electron detector which produces an image of the surface of the specimen, and a back-scattered electron detector which produces an image whose pixel values correspond to the average atomic number (Z) of a given material.

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 SEM-EDS Bruker Quick Start Guide.

For additional resources, 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

Turning on the SEM

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

1. .
2. 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

1. 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.

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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.

2. Important: use compressed air to

1. blow off any loose material on the sample

1. 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

1. yellow

1. .
2. In the Nanoeye software, click the “Sample Information” text box (Figure 4, red rectangle), enter the height and width values, then press Enter.

1.  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]

1. . 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 (+/-

1. 1. 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

1. 1. .

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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

1. voltage 

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   Figure 7. Operational start-up window

   
   

2. 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.

1. 1. 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.
2. Click the START button to generate

1. the electron beam (Figure 7). 
2. 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.

1. The focal plane should generally be at approximately

1. 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.

2. 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)

1. . 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.

Recommended settings

D. Capture an image

Annotating the image (optional)

Saving the image

File structure (need to save to C:\SEM)

E. Shutdown

1. When you are finished scanning, turn the beam off by clicking the Stop button in the Operation dropdown panel.
2. Bring the motor back to the zero position by clicking Home
3. Nanoeye software can now be closed
4. Push the vacuum button (Figure 1.3). This will initiate the chamber filling with air, and you can track the progress using the LED strip on the front of the instrument.
5. The door will be easily opened once pumped with air
6. Remove your specimen mount from the holder (Figure 5), removing the specimen from the stub

1. adapter if used.
2. Close the door, then push the vacuum button (Figure 1.3) to initiate a vacuum sequence.
3. Once the vacuum has be reached, turn off the SEM (Figure 1.2). When not in use, the SEM should be closed, at vacuum, and powered off.

III. Uploading Data to LIMS

The SEMUploader application is used to format the tests for uploading to LIMS

. It is available through the IODPLauncher application.

1. Select that sample you used to take the test. (You may also provide the sample's Text ID, if you have it)
   
   1. If uploading EDS data to a SEM test that was already uploaded, select the 'Existing SEM' tab.
2. When done selecting the sample, click 'Select' button.
   1. If uploading EDS data to an existing SEM test, select the SEM image you would like to upload EDS data for.
3. Enter the comments and initials and click 'Done' button.
4. Add as many SEM tests for the selected sample as you need.
5. You may edit records by going to the Records tab.
6. Make a folder for the all the EDS data you would like to upload. You may upload multiple EDS folders to a single SEM test.
   1. It is strongly suggested that the user and technicians upload to LIMS all formats available for the EDS analysis (Mapping mode - .bcf; Line Scan mode - .rtl; Objects mode - .rto; spectra for each mode - .spx; ESPRIT Compact standard Report - .rpt; Microsoft Word - .doc);
7. When finished adding all the SEM and EDS data for the selected sample, clicked the 'Finish and upload' button.
8. This will create a file in the C:\data\in folder (and any other folder you may add under the Settings tab)
9. Use the MUT application to upload this file. (Make sure MUT is configured to use C:\data\in and SEM is an active analysis)

IV. Credits

Version 1.0 of this document was written by Luan Heywood

and Kara Vadman, on Exp 397T

, with previous contributions by Sarah Kachovich.

V. Archived Versions