red = needs attention

italics = drafty language

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

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

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 SEC SEM with Bruker EDS Quick Start Guide.

What are additional resources (link them here)

Scheme of a SEM with SE, BSE and EDS volume of interaction and detectors.

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 
  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.

Figure 2. Nanoeye software icon

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.

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. 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. IMAGE GOES HERE 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.
    3. 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.

Figure 5. Mounting the stub to the stage.

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

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

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). IMAGE GOES HERE 

  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 

    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. 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.
  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. how to change the filament

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.

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.

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

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

Advanced image correction (for technicians)

Correct the beam shape

Align the strip aperture

Adjust the stigmation

Align the beam

Recommended settings

SampleSample preparationSEM settings
Foraminifera or other large (50 um - 1 mm) particles of geological materialPicked and mounted on an SEM stub, optionally sputter coated
  • 1-5 kV (higher if sample is Au/Pd coated)
Diatoms or nannofossilsSmear slide, sputter coated with Au/Pd
  • 5-10 kV
Polished thin section/polished thick sectionPolished thin/thick section placed in thin section holder, fastened down using the clips and carbon tape, carbon coated
  • 10-20 kV
Larger amounts of geological material, prepared as a polished grain mountGrain mount placed in grain mount holder, carbon coated
  • 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

  • Make the spot size more diffuse
  • Decrease the accelerating voltage
  • Try Low Vacuum
  • Make sure the sample is conductive

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 adapter if used.
  7. Close the door, then push the vacuum button (Figure 1.3) to initiate a vacuum sequence.
  8. 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

Updated SEM Uploader

IV. Preventative Maintenance

Once per expedition

  • Using a small extendable mirror and a flashlight, carefully examine the mylar cap on the tip of the EDS detector to make sure that it is still intact. If it is broken (typically due to collision), take a picture and send it to the SEM vendor for instructions on how to replace the mylar cap.
  • Examine the stage and use canned air to blow off any dust present on the stage. The chamber should not require cleaning, but if there is any material or fingerprints on the inside, they can be cleaned off using isopropanol and a kimwipe.
  • Examine the sealing surface on the SEM door for any accumulated dust, and clean off if necessary. A small amount of vacuum grease (jar in SEM accessories tote, or Dow-Corning 111) can be applied to the sealing surface.
  • Examine the oil level on the vacuum pumps and add more/replace if necessary. The vacuum pump on the SEC SEM takes special high-vacuum oil, which should not be substituted with other vacuum oils.

1-2 times per year

  • Go into the SEM software and check the number of hours on the filament. Filaments typically last around ___ hours, and a failing filament is typically indicated by the emission current reading in the SEM software (any values outside 110+/-20 uA). It is good to be aware of where a filament is, relative to its livespan, so technicians can be prepared to replace it.
  • Drain the oil from the vacuum pumps and replace with new oil. The vacuum pump on the SEC SEM takes special high-vacuum oil, which should not be substituted with other vacuum oils.

IV. Credits

Version 1 of this document was written by Luan Heywood and Kara Vadman, on Exp 397T, with previous contributions by Sarah Kachovich. This document was reviewed by ___ on ___.

V. Archived Versions

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