Introduction

The TM3000 Scanning Electron Microscope (SEM) captures surface images of a specimen in a vacuum chamber by scanning an electron beam over a sample. 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 bombard the specimen and secondary and backscattered electrons are formed. These electrons are detected by a secondary electron detector which produces an image of the surface of the specimen. Figure 1 below i from Hitachi's 'Let us Familiarize Ourselves with the SEM!' document to illustrates generally how a SEM works. For more information regarding how a SEM works refer to SEM Vendor Manual and Let Us Familiarize Ourselves with SEM!.

Figure 1. Image taken from 'Let Us Familiarize Ourselves with the SEM!' by Hitachi. Illustrates general parts and introduction to how SEM produces an image.

Apparatus and Materials

Instrument Set Up

Start Microscope

1.On the back of the instrument make sure the main power breaker is in the 'On' position

2. Flip the 'On' switch on the right side of the microscope

3.The SEM will flash the yellow 'Air' and blue 'Evac' status indicator lights and then resume the last operation it was under when turned off.

Sample Preparation

Samples can be imaged untreated, sputter coated, or with silver paint. The purpose of treating the sample is to reduce the charge on the sample. Charge on the sample attenuates the electron beam and produces fuzzy images that make it difficult to identify structures or features on the sample. It is recommended to first image an untreated sample and if too much charge is detected in the image, apply a treatment and then re-image.

All Samples

A sample can be loaded into the SEM in a variety of ways. Three things are needed to assemble a sample mount are listed below:

1.A mount to put in the SEM

2. A piece of adhesive to stick to the stub and to a sample

3. Sample material or glass containing a sample

The tables below list the various options of how to assemble a mount.

Once the sample has been mounted you may proceed to Loading Sample or to Additional Sample Treatments.

Additional Sample Treatments

Sputter Coating Treatment

Introduction

The Leica EM ACE200 coating system is used for precise coating of samples for subsequent examination with a scanning electron microscope (SEM). Samples are metal coated using the sputtering method where argon plasma erodes a target material. Carbon coating is achieved by carbon thread evaporation. Any material can be processed as long as it is not sensitive to vacuum, argon plasma, or the heat generated during carbon coating. Sputter coating prevents charging the specimen with an electron beam in the SEM high-voltage mode. Metal coatings are also useful for increasing signal-to-noise ratio. The ACE200 can be configured as a sputter coater, carbon coater, or both, where the two processes can be switched over from one to the other.

With the Hitachi TM3000 SEM, sputter coating is not required for all samples. Images up to a magnification of 4000x are of high quality without any sputter coating in Charge-up Reduction Mode (Settings > Observation Mode).

For detailed observations in magnifications greater than 4000x and for very delicate fossils, sputter coating before using the SEM can be used to potentially receive higher quality images.

Possible Targets

• Gold Palladium
• Carbon Thread
• The instrument can use other metal targets but these are currently not supplied to the ship

Argon Gas Supply

The working gas (argon) must be supplied under a pressure of ~500 mbar ±100 mbar (5.6—8.4 psi above atmosphere). The gas should be at least 99.99% pure.

Caution! Do not use a standard two-stage regulator for the sputter coater unless it is equipped with a low-pressure delivery-side gauge. Most regulators don’t effectively pick up pressure until 10 or even 15 psi or higher. It may be necessary to put a second low-pressure-range regulator downline from the main line regulator. Make sure you have the right tool for the job!

Sputtering a Sample

Set Up Parameters

• Target material: select from a drop-down list; the instrument automatically chooses parameter settings.
• Directional mode: intended for relatively flat samples; base vacuum is 4 × 10^–2 mbar for this mode.
• Diffuse mode: intended for more topographic samples; base vacuum is 8 × 10^–2 mbar for this mode.
• Sputter current/purge cycles: automatically set when target is selected. Change under the Setup button.
• Process termination time: select the Clock icon then use the +/– buttons to select a time in seconds.
• Rotation speed: press the Rotation button, check Rotation On, and use the +/– buttons to set the speed.

Starting Measurement

1.Check the Argon levels on the regulator and confirm the level is between 5.6 - 8.4 psi

2. Wearing gloves, place sample(s) on stage. Do not put sample(s) in the center of the stage or material will not land on the sample.

3. Turn on the vacuum pump behind the Sputter Coater

4. Turn on the main power supply to the instrument and make sure the chamber door and cover are closed.

5. Select the Sputtering button.

6. Press the Pump button to start pump-down.

7. During pump-down, set the process parameters:

• Target material (last material used is presented by default)
• Mode (Directional or Diffuse) button
• Sputter current/purge cycles (preset when target was selected)
• Process termination time (e.g., 12 seconds)
• Rotation speed (e.g., 1)

8. Press the Start button (which then becomes the Stop button). The Start button can be pressed while the Sputter Coater is still achieving vacuum. Below is the sputtering sequence:

• Pump until base vacuum is reached.
• Stabilize the plasma.
• Pre-sputter, if target requires it (to clean oxidation from the target and to enable a stable sputter rate).
• Start the sputter processing by opening the shutter and starting rotation (if activated).
• Terminate sputtering by time.
• Close the shutter.
• Display results of the process.
• Vent or stay under vacuum, per settings.

9. When sputtering complete press Vent to release the vacuum. Then the door will open and the samples can be removed. 

Note: if the vacuum cannot stabilize, make sure the argon supply valve is open and pressure is on the line. What happens when pump can't stabilize?

Silver Paint Treatment

Silver paint allows a path for electrons to discharge from the sample.

Prepare Sample

1.Dab a very small amount of paint on the side of sample and extending to the edge of the sample stub. The painted sample area will obscure surface features so it is important to use very little on the sample. 

Loading Sample in SEM

Screw on Stub

Adjust Height of Sample Assembly

1.Make sure the Air status bar is solid yellow. This indicates the chamber is not in a vacuum state and the chamber can be opened. If not, press the Evac/Air button to infill the chamber with air. During the fill process the status bar will flash yellow. When the chamber is full of air and safe to open, the staus bar will change to a solid yellow.

2. Open the door.

3. Using the red Hex Key, turn the set screw about half a turn to loosen the sample stage. Do not unscrew too much or the set screw could fall out.

4. Pull out the sample stage.

5. Place the sample stage in the black Specimen Height Adjuster tool (Fig XX).

6. The goal is to have the stub/sample just below the black bar. This bar represents the height of the door seal. If the stub is touching the black bar on the height adjuster then it will hit the door seal. Unscrew the larger nut and adjust the height. Retighten by tightening the smaller nut. 

Image Added

Put in SEM

1.Open the door to the vacuum chamber

2. Put the sample assembly back in the hole.

3. Tighten the set screw using the hex key. Hand Tight is sufficient.

Image Added

4. Look through the v plate in the chamber. With the knobs X and Y Stage Knobs on the SEM door, adjust the sample stage position so the cross-hairs on the stage aligns in the V.

Image Added

4. Close the door.

5. Push the Evac/Air button to put the chamber into a vacuum. Press on the door for a few moments to help get the vacuum started. It will take several minutes to get the chamber down to vacuum.

Capture Image

While the SEM is drawing down the vacuum the image capture software can be opened.

Open Software

1.Click the TM3000 software icon on the desktop

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2. Start up screen will open. Note the screen will appear black at first. The electron beam gun is not activated until a user presses Start.

Image Added

Below are the tools available in the software:

• Start: Turns on the high voltage which generates an electron beam. Must press start to generate an image.
• Fast: Image updates quickly. Useful to select 'Fast' while changing Field of View.
• Slow: Image updates slowly. If in calm sea state, can produce a higher quality image. 
• Reduce: 
• Freeze: Stops updating the image and preserves the image as it was at the time of pressing Freeze. 
• Magnification: Control to increase or decrease magnification. Ranges from x40 to x30,000.
• Auto B/C: Click this to automatically adjust the brightness and contrast of the image.
• Auto Focus: Click this to automatically focus the image. 
• Image Mode: 
• 5 kV / 15 kV / Analy:
  
• Quick Save:
  
• Save:

3. Press Start. 

Uploading Data

Data upload requires a two softwares used in the order below:

1. SEMUploader to convert files into a format readable in the database
2. MegaUploaderTron (MUT) to upload files into the database.

Two file types are generated by the SEM:

• .tif : This file is the image taken by the software.
• .txt : This file contains the metadata for the image. This file includes parameters under which the image was taken, instrument specifics, filename, timestamp, and directory.

The SEMUploader uses these two files to generate a new .SEM file. This is the only file uploaded to LORE and contains callouts to the .tif and .txt file.

SEMUploader

1.Open SEMUploader (Figure XX)

2. The Main Window opens up (Figure XX). Click Select Image in the upper left corner.

3. Navigate to the images and select the desired .tif file.

4. The image populates the screen. Click Process to move forward. OR click Exit if you decide to upload later. CLEAR will clear out the image from the selection. The user can select another image right away if the one chosen is not the one that they wanted to upload.

At this point a user could encounter two potential errors:

• If the user selects a file that is not a .tiff file the message below will appear (Fig XX). To resolve click on the appropriate file type
  
  
  
  
• If the user selects a file that does not have a corresponding .txt file the message below will appear (Fig XX)

5. After clicking Process the sample information window appears. In this window we can assign a sample to the image and text files.

MUT Upload

1.Click on the MUT icon to open the software

2. Login to MUT with your LIMS applications username and password.

3. The files should populate when MUT opens.

4. Click Upload. 

The software can be left open in the background with the Automatic Upload box checked.

LIMS Integration

Images and metadata can be accessed in LORE.

1.Open LORE in a web browser

2. On the left side of the screen expand Images > Scanning Electron Microscope > select either Standard or Expanded (Figure XX).

Below is a table describing the information uploaded to LORE.

Maintenance

Sputter Coater

Switching Target Sources

To switch between carbon thread and sputter coating, exchange the source (sputter or carbon thread) and choose the process on the main screen of the instrument. The instrument automatically detects if the correct source is installed and gives and error message if it does not match the selected process.

Loading Gold Palladium Target

There is an ACE200 youtube video that shows the loading of the target at http://www.youtube.com/watch?v=Kd1lyKIDT8.

The procedure is as follows:

1. Wear gloves and make sure the instrument is fully vented.
2. Open the source cover and unplug the connectors.
3. Unscrew the 2 sputter head screws to remove the flange.
4. Remove the bayonet ring by turning and insert the sputter target as shown in Figure 2, below.
5. Fix the target by gently tightening the bayonet ring; tighten by hand only.
6. Replace the sputter head and gently tighten the fastening screws.
7. Connect the cables and close the cover.

Loading Carbon Thread

The carbon thread can be loaded as a single thread or as a double thread. Thin layers from 1 to at least 20 nm can be achieved (there is variance in threads).

To minimize carbon thread waste, when loading a double thread, cut a piece of thread twice as long as the width of the black door frames of the coater. Fold the thread into half and load it as follows:

1. Before beginning, prepare the following on a clean surface: gloves, carbon head, Torx TX 10 key, brush, carbon thread.
2. Make sure the instrument is vented. Wear gloves.
3. Open the source cover and unplug connectors.
4. Unscrew the 2 evaporation head screws to remove the flange.
5. Loosen all 5 clamp screws with the Torx key.
6. Remove any carbon residue using the brush.
7. Loop the carbon thread around the first clamp; pull both ends gently to the left while tightening the screw.
8. Wind the thread around the other clamps, taking care that the thread slides into the clamping groove.
9. Pull the thread gently and tighten the last clamp.
10. Tighten the 3 remaining screws and trim thread on both ends as shown in Figure 3, below.
11. Replace the head, gently tighten the fastening screws, connect the cables, and close the cover.

Also note that there is an ACE200 youtube video where the loading of the thread can be seen at http://www.youtube.com/watch?v=yKd1IyKIDT8).